Compositions and methods for stabilizing transthyretin and inhibiting transthyretin misfolding

ABSTRACT

Kinetic stabilization of the native state of transthyretin is an effective mechanism for preventing protein misfolding. Because transthyretin misfolding plays an important role in transthyretin amyloid diseases, inhibiting such misfolding can be used as an effective treatment or prophylaxis for such diseases. Treatment methods, screening methods, as well as specific transthyretin stabilizing compounds are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of co-pending U.S. application Ser.No. 10/741,649, filed Dec. 19, 2003, entitled “COMPOSITIONS AND METHODSFOR STABILIZING TRANSTHYRETIN AND INHIBITING TRANSTHYRETIN MISFOLDING.”U.S. application Ser. No. 10/741,649 claims priority from U.S.Provisional Application No. 60/435,079, filed Dec. 19, 2002, and U.S.Provisional Application No. 60/465/435, filed Apr. 24, 2003. The entiredisclosures of the above-referenced applications are incorporated hereinby reference in their entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funds used to support some of the studies disclosed herein were providedby grant number NIH DK 46335 awarded by the National Institutes ofHealth. The Government may have certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to protein misfolding. Moreparticularly, this invention provides compositions and methods forstabilizing transthyretin, inhibiting transthyretin misfolding, andtreating amyloid diseases associated thereto.

BACKGROUND OF THE INVENTION

Transthyretin (TTR) is a 55 kDa homotetrameric protein present in serumand cerebral spinal fluid. The function of TTR is to transportL-thyroxine (T₄) and holo-retinol binding protein (RBP). TTR is one ofgreater than 20 nonhomologous amyloidogenic proteins that can betransformed into fibrils and other aggregates leading to diseasepathology in humans. These diseases do not appear to be caused by lossof function due to protein aggregation. Instead, aggregation appears tocause neuronal/cellular dysfunction by a mechanism that is not yetclear.

Under denaturing conditions, rate limiting wild type TTR tetramerdissociation and rapid monomer misfolding enables misassembly intoamyloid, putatively causing senile systemic amyloidosis (SSA).Dissociation and misfolding of one of more than eighty TTR variantsresults in familial amyloid polyneuropathy (FAP) and familial amyloidcardiomyopathy (FAC).

The TTR tetramer has two C₂ symmetric T₄-binding sites. Negativelycooperative binding of T₄ is known to stabilize the TTR tetramer andinhibit amyloid fibril formation. Unfortunately, less than 1% of TTR hasT₄ bound to it in the human serum, because thyroid-binding globulin(TBG) has an order of magnitude higher affinity for T₄ in comparison toTTR. Furthermore, the serum concentration of T₄ is relatively low (0.1μM) compared to that of TTR (3.6–7.2 μM).

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery that kineticstabilization of the native state of transthyretin inhibits proteinmisfolding. This discovery is important because of the role that proteinmisfolding plays in a variety of disease processes, includingtransthyretin amyloid diseases. By inhibiting transthryetin misfolding,one can intervene in such a disease, ameliorate symptoms, and/or in somecases prevent or cure the disease.

The discovery that kinetic stabilization of the native state oftransthyretin effectively inhibits misfolding allows for the developmentof therapeutic compositions with potentially high specificity and lowtoxicity. Thus, although exemplary biaryl reagents which have theability to stabilize transthryetin are disclosed herein, one can designother reagents which selectively stabilize the protein. For example, asdescribed herein, it is possible to design and prepare polychlorinatedbiphenyls, diflunisal analogs, or benzoxazoles that highly selective forbinding to transthyretin and that stabilize the native state oftransthyretin.

In on aspect, the invention features a method of screening for acompound that prevents or reduces dissociation of a transthyretintetramer. The method can include the following steps: contacting atransthyretin tetramer with a candidate compound; and determiningwhether the candidate compound increases the activation energyassociated with dissociation of the transthyretin tetramer, therebypreventing or reducing dissociation of the transthyretin tetramer. Themethod can optionally include an additional step of measuring theability of the candidate compound to inhibit fibril formation.

In one embodiment, the method includes a step of determining whether thecompound prevents dissociation of the transthyretin tetramer bydestabilizing the dissociation transition state of the transthyretintetramer. In another embodiment, the method includes a step ofdetermining whether the compound prevents dissociation of thetransthyretin tetramer by stabilization of the transthyretin tetramermore than the dissociative transition state.

The candidate compound used in such a method can optionally be a smallmolecule. Such a small molecule can stabilize the native state oftransthyretin through tetramer binding, thereby slowing dissociation andamyloidosis under denaturing and physiological conditions through akinetic stabilization mechanism. The compound optionally exhibitsbinding stoichiometry exceeding 0.1 to TTR in human blood whenadministered at a concentration of 10.6:M.

A small molecule can optionally have a molecular weight of less than1500 and bind to transthyretin non- or positively cooperatively andimpart a binding energy of >2.3 kcal/mol. The small molecule can exhibitK_(d1) and K_(d2)<100 nM (e.g., <10 nM) and/or a high plasmaconcentration, which both contribute to protein stabilization exceeding2.0 kcal/mol. The small molecule can also decrease the yield ofamyloidosis and decrease the rate of acid-mediated or MeOH mediatedamyloidogenesis and/or decrease the rate of urea mediated TTRdissociation.

In some embodiments, the small molecule includes biphenyl amines,biphenyls, oxime ethers, benzazoles or other structures composed of twoaromatic rings where one bears hydrophilic groups such as an acid or aphenol and the other bears hydrophobic groups such as halogens oralkyls.

In one embodiment, the candidate compound is a biaryl where one ringbears a hydrophilic substituent(s) and the other has hydrophobicsubstituents or a biaryl where both rings bear at least one hydrophilicsubstituent. The hydrophilic group can be a phenol, a COOH, a benzylalcohol, a boronic acid or ester, a tetrazole, an aldehyde or a hydratedaldehyde or a functional group that serves as either a H-bond donor oracceptor to the protein directly or through a water mediated H-bond. Thebiaryl can be a symmetrical biaryl having both rings substituted withhydrophilic functionality including phenols, carboxylates and alcoholsand in some cases halogens to compliment the halogen binding pockets inTTR, e.g., a biaryl with the following functionality 3-Cl, 4-OH, 5-Cland 3′-Cl, 4′-OH, 5′-Cl. In one embodiment, at least one ring of thebiaryl is substituted with 2,4-difluoro or 3,5-difluoro or 2,6-difluoroor 3,5-dichloro or 3-Cl, 4-OH, 5-Cl or 3-F, 4-OH, 5-F, 3-COOH, 4-OH or3-OH or 3-COOH or 4-COOH or 3-CH2OH or 4-CH2OH substituents. Anexemplary biaryl is a polychlorinated biphenyl, e.g., a hydroxylatedpolychlorinated biphenyl wherein at least one ring one bears OH and/orCl substituents including 3-Cl, 4-OH, 5-Cl or 2-Cl, 3-Cl, 4-OH, 5-Cl or3,4-DiCl, or 2,3,4-trichloro or 2,3,4,5-tetrachloro. Halogens other thanchloride can be used in the candidate compound. The candidate compoundcan be a benzoxazole.

In one embodiment, the candidate compound is a diflunisal analog. Thestructure of diflunisal as well as a variety of diflunisal analogs aredescribed herein. The diflunisal analog can optionally have reduced orabsent NSAID activity as compared to diflunisal. For example, thediflunisal analog can have reduced or absent cyclooxygenase inhibitoractivity as compared to diflunisal.

In one embodiment, the method includes an additional step of determiningwhether the diflunisal analog exhibits NSAID activity. For example, themethod can include a step of determining whether the diflunisal analogexhibits cyclooxygenase inhibitor activity.

The transthyretin used in the screening methods can be wild typetransthyretin or a mutant transthyretin, such as a naturally occurringmutant transthyretin causally associated with the incidence of atransthyretin amyloid disease such as familial amyloid polyneuropathy orfamilial amyloid cardiomyopathy. Exemplary naturally occurring mutanttransthyretins include, but are not limited to, V122I, V30M, L55P (themutant nomenclature describes the substitution at a recited amino acidposition, relative to the wild type; see, e.g., Saraiva et al. (2001)Hum. Mut. 17:493–503).

The invention also provides for methods for the stabilization oftransthyretin in a tissue or in a biological fluid, and therebyinhibiting misfolding. Generally, the method comprises administering tothe tissue or biological fluid a composition comprising a stabilizingamount of a compound described herein that binds to transthyretin andprevents dissociation of the transthyretin tetramer by kineticstabilization of the native state of the transthyretin tetramer.

Thus, methods which stabilize transthyretin in a diseased tissueameliorate misfolding and lessen symptoms of an associated disease and,depending upon the disease, can contribute to cure of the disease. Theinvention contemplates inhibition of transthyretin misfolding in atissue and/or within a cell. The extent of misfolding, and therefore theextent of inhibition achieved by the present methods, can be evaluatedby a variety of methods, such as are described in the Examples.

Accordingly, in another aspect the invention includes a method oftreating a transthyretin amyloid disease, the method comprisingadministering to a subject diagnosed as having a transthyretin amyloiddisease a therapeutically effective amount of a compound that preventsdissociation of a transthyretin tetramer by kinetic stabilization of thenative state of the transthyretin tetramer.

In one embodiment, the invention features a method of treating atransthyretin amyloid disease, the method comprising administering to asubject diagnosed as having a transthyretin amyloid disease atherapeutically effective amount of a diflunisal analog (e.g., adiflunisal analog that prevents dissociation of a transthyretintetramer) that prevents dissociation of a transthyretin tetramer. Thediflunisal analog can optionally have reduced or absent NSAID activity(e.g., cyclooxygenase inhibitor activity) as compared to diflunisal.

In another embodiment, the invention features a method of treating atransthyretin amyloid disease, the method comprising administering to asubject diagnosed as having a transthyretin amyloid disease atherapeutically effective amount of a polychlorinated biphenyl (e.g., apolychlorinated biphenyl that prevents dissociation of a transthyretintetramer) that prevents dissociation of a transthyretin tetramer. Thepolychlorinated biphenyl can be a hydroxylated polychlorinated biphenyl.

In another embodiment, the invention features a method of treating atransthyretin amyloid disease, the method comprising administering to asubject diagnosed as having a transthyretin amyloid disease atherapeutically effective amount of a benzoxazole (e.g., a benzoxazolethat prevents dissociation of a transthyretin tetramer) that preventsdissociation of a transthyretin tetramer.

The transthyretin amyloid disease can be, for example, familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, or senile systemicamyloidosis.

The subject treated in the present methods can be a human subject,although it is to be understood that the principles of the inventionindicate that the invention is effective with respect to all mammals. Inthis context, a “mammal” is understood to include any mammalian speciesin which treatment of diseases associated with transthyretin misfoldingis desirable, particularly agricultural and domestic mammalian species.

The compounds described herein (e.g., biaryl compounds such asdiflunisal analogs, polychlorinated biphenyls, or benzoxazoles) can beformulated with a pharmaceutically acceptable to prepare apharmaceutical composition comprising the compound. As used herein, theterms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects.

The invention also encompasses the use of any of the compounds orpharmaceutical compositions described herein for the treatment of atransthyretin amyloid disease (e.g., familial amyloid polyneuropathy,familial amyloid cardiomyopathy, or senile systemic amyloidosis).

The invention also encompasses the use of any of the compounds orpharmaceutical compositions described herein in the manufacture of amedicament for the treatment of a transthyretin amyloid disease (e.g.,familial amyloid polyneuropathy, familial amyloid cardiomyopathy, orsenile systemic amyloidosis).

The compounds and treatment methods described herein provide significantadvantages over the treatments options currently available for TTRamyloidosis. TTR amyloidosis typically leads to death in ten years, anduntil recently, was considered incurable. Liver transplantation is aneffective means of replacing the disease-associated allele by a WTallele in familial cases because the liver is typically the source ofamyloidogenic TTR. While liver transplantation is effective as a form ofgene therapy it is not without its problems. Transplantation iscomplicated by the need for invasive surgery for both the recipient andthe donor, long-term post-transplantation immunosuppressive therapy, ashortage of donors, its high cost, and the large number of TTRamyloidosis patients that are not good candidates because of theirdisease progression. Unfortunately, cardiac amyloidosis progresses insome familial patients even after liver transplantation because WT TTRoften continues to deposit. Nor is central nervous system (CNS)deposition of TTR relieved by transplantation owing to its synthesis bythe choroid plexus. Transplantation is not a viable option for the mostprevalent TTR disease, senile systemic amyloidosis (SSA), affectingapproximately 25% of those over 80 due to the deposition of WT TTR.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting the T4 binding site oftransthyretin.

FIGS. 2A and 2B are graphs depicting the time course of transthyretinunfolding in the presence of different inhibitors.

FIGS. 3A and 3B are graphs depicting the time course of fibril formationin the presence of different inhibitors.

FIGS. 4A and 4B are graphs depicting the time course of fibril formationin the presence of different inhibitors.

FIG. 5 depicts the structures of polychlorinated biphenyls screened forbinding to transthyretin in blood plasma.

FIG. 6 depicts the structures of hydroxylated polychlorinated biphenylswhose binding to transthyretin in plasma was evaluated along with theiramyloid fibril inhibition properties in vitro.

FIG. 7 is a graph depicting suppression of transthyretin fibrilformation by benzoxazole compounds. The position of the carboxyl on thebenzoxazole is shown along the left-hand side, while the C(2) phenylring is shown along the bottom. The bars indicate the percent fibrilformation (ff), that is, the amount of fibrils formed from transthyretin(3.6 μM) in the presence of the benzoxazole compound (7.2 μM) relativeto the amount formed by transthyretin in the absence of inhibitor (whichis defined as 100%).

FIG. 8 is a graph depicting stoichiometry (s) of benzoxazoles bound totransthyretin after incubation in human blood plasma.Immunoprecipitation with a resin-bound antibody was used to capturetransthyretin. Following release of transthyretin from the resin, theamounts of transthyretin and inhibitor were quantified from the areasunder their peaks in an HPLC chromatogram. The maximum possible value ofs is 2. Compound numbers are shown along the bottom axis. The thinvertical lines indicate the measurement error.

FIG. 9 is a graph depicting dissociation as a function of time (t) forwt transthyretin (1.8 μM) in 6 M urea without inhibitor, or in thepresence of 3.6 μM of compounds 20, 21, or 27, or 1.8 μM compound 20.

FIG. 10 depicts the X-ray co-crystal structure of compound 20 bound totransthyretin. Equivalent residues in different subunits aredistinguished with primed and unprimed residue numbers, as are the pairsof halogen binding pockets.

DETAILED DESCRIPTION OF THE INVENTION

At least some amyloid diseases appear to be caused by the deposition ofany one of more than 20 nonhomologous proteins or protein fragments,ultimately affording a fibrillar cross-θ-sheet quaternary structure.Formation of amyloid fibrils from a normally folded protein liketransthyretin requires protein misfolding to produce anassembly-competent intermediate. The process of transthyretin (TTR)amyloidogenesis appears to cause three different amyloid diseases—senilesystemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) andfamilial amyloid cardiomyopathy (FAC). SSA is associated with thedeposition of wild-type TTR, while FAP and FAC are caused by theamyloidogenesis of one of over 80 TTR variants. See, for example, Colon,W.; Kelly, J. W. Biochemistry 1992, 31, 8654–60; Kelly, J. W. Curr.Opin. Struct. Biol. 1996, 6, 11–7; Liu, K.; et al. Nat. Struct. Biol.2000, 7, 754–7; Westermark, P.; et al. Proc. Natl. Acad. Sci. U.S.A.1990, 87, 2843–5; Saraiva, M. J.; et al. J. Clin. Invest. 1985, 76,2171–7; Jacobson, D. R.; et al. N. Engl. J. Med. 1997, 336, 466–73;Buxbaum, J. N.; Tagoe, C. E. Ann. Rev. Med. 2000, 51, 543–569; andSaraiva, M. J. Hum. Mutat. 1995, 5, 191–6, each of which is incorporatedby reference in its entirety.

TTR is a 55 kDa homotetramer characterized by 2,2,2 symmetry, having twoidentical funnel-shaped binding sites at the dimer-dimer interface,where thyroid hormone (T4) can bind in blood plasma and CSF. TTR istypically bound to less than 1 equiv of holo retinol binding protein.TTR misfolding including tetramer dissociation into monomers followed bytertiary structural changes within the monomer render the proteincapable of misassembly, ultimately affording amyloid. The availabletreatment for FAP employs gene therapy mediated by liver transplantationto replace variant TTR in the blood with the wild type (WT) protein.This approach will likely not be effective for FAC due to the continueddeposition of WT TTR, nor would it be useful for the treatment of SSA,where the process of WT TTR deposition appears to be causative. Livertransplantation therapy would also fail for approximately 10 of the TTRvariants that deposit amyloid fibrils in the leptomeninges leading toCNS disease, as this TTR is synthesized by the choroid plexus. Hence, itis desirable to develop a general noninvasive drug-based therapeuticstrategy. It can be desirable for the drug to be non-protein,non-peptide, or non-nucleic acid based. See, for example, Blake, C. C.;et al. J. Mol. Biol. 1978, 121, 339–56; Wojtczak, A.; et al. ActaCrystallogr., Sect. D 1996, 758–810; Monaco, H. L.; Rizzi, M.; Coda, A.Science 1995, 268, 1039–41; Lai, Z.; Colon, W.; Kelly, J. W.Biochemistry 1996, 35, 6470–82; Holmgren, G.; et al. Lancet 1993, 341,1113–6; Suhr, O. B.; Ericzon, B. G.; Friman, S. Liver Transpl. 2002, 8,787–94; Dubrey, S. W.; et al. Transplantation 1997, 64, 74–80; Yazaki,M.; et al. Biochem. Biophys. Res. Commun. 2000, 274, 702–6; andCornwell, C. G. III; et al. Am. J. of Med. 1983, 75, 618–623, each ofwhich is incorporated by reference in its entirety.

Synthesis of Diflunisal Analogs that Inhibit Transthyretin AmyloidFibril Formation

TTR misfolding leading to amyloid fibril formation can be prevented byT4-mediated stabilization of the tetramer. Several structurally diversefamilies of tetramer stabilizers bind to one or both T4 sites within TTRand prevent amyloidosis without the likely side effects of the hormoneT4. These tetramer stabilizing compounds include several non-steroidalanti-inflammatory drugs (NSAIDS) such as flufenamic acid, diclofenac,flurbiprofen and diflunisal, that appear to function by increasing thekinetic barrier associated with tetramer dissociation throughground-state binding and stabilization. Because TTR is the secondarycarrier of T4 in blood plasma, greater than 95% of TTR's T4 bindingcapacity remains unutilized, allowing for administration of tetramerstabilizing compound that target these sites. Because diflunisal is acyclooxygenase-2 inhibitor long-term administration could lead togastrointestinal side effects. Analogs of diflunisal that have reducedor absent NSAID activity, but possess high affinity for TTR in bloodplasma, are therefore desirable. The first step toward this goal is thedesign and synthesis of diflunisal analogs as inhibitors of amyloidfibril formation. See, for example, Miroy, G. J.; et al. Proc. Natl.Acad. Sci. U.S.A. 1996, 93, 15051–6; Klabunde, T.; et al. Nat. Struct.Biol. 2000, 7, 312–21; Baures, P. W.; Peterson, S. A.; Kelly, J. W.Bioorg. Med. Chem. 1998, 6, 1389–401; Petrassi, H. M.; et al. J. Am.Chem. Soc. 2000, 122, 2178–2192; Baures, P. W.; et al. Bioorg. Med.Chem. 1999, 7, 1339–47; Sacchettini, J. C.; Kelly, J. W. Nat. Rev. DrugDisc. 2002, 1, 267–275; Oza, V. B.; et al. J. Med. Chem. 2002, 45,321–32; Bartalena, L.; Robbins, J. Clin. Lab. Med. 1993, 13, 583–98;Aldred, A. R.; Brack, C. M.; Schreiber, G. Comp. Biochem. Physiol. BBiochem. Mol. Biol. 1995, 111, 1–15; and Mao, H. Y.; et al. J. Am. Chem.Soc. 2001, 123, 10429–10435, each of which is incorporated by referencein its entirety.

The subunits of the TTR tetramer are related by three perpendicularC₂-axes. FIG. 1 is a schematic representation of the T4 binding site ofTTR, demonstrating the forward binding mode where the inhibitorcarboxylate participates in electrostatic interactions with theM-ammonium of Lys 15 and 15′. The two equivalent T4 binding sitescreated by the quaternary structural interface are interchanged by thetwo C₂ axes that are perpendicular to the crystallographic C₂ axis ofsymmetry. Each T4 binding site can be divided into an inner and outerbinding cavity. See, for example, Blake, C. C.; Oatley, S. J. Nature1977, 268, 115–20, which is incorporated by reference in its entirety.The inner binding cavity comprises a pair of halogen binding pockets(HBP), designated HBP 3 and 3′, made up by the side chains of Leu 17,Ala 108, Val 121, and Thr 119. The convergence of four Ser 117 sidechains from each subunit defines the innermost region and interfacebetween the two identical binding sites. The Ser 117 hydroxyl groups canserve as hydrogen bond donors or acceptors to complimentaryfunctionality on the compound (e.g., an inhibitor of amyloid formation)or mediate electrostatic interactions with the compound through watermolecules. The outer binding site is composed of HBP 1 and 1′, while HBP2 and 2′ are positioned at the interface of the inner and outer bindingcavities. The Lys 15 and 15′ M-ammonium groups define the very outerreaches of the outer binding cavity, allowing for electrostaticinteractions with anionic substituents on a compound. Many of the TTRtetramer stabilizing compounds bind in the forward binding mode, wherean anionic substituent on the hydrophilic phenyl ring positioned in theouter binding pocket engages in an electrostatic interaction with theLys 15 M-ammonium groups. In the forward binding mode, a hydrophobicphenyl ring (often substituted with halogens) can occupy the innerbinding pocket. Examples of binding in the opposite orientation (thereverse binding mode), however, have also been observed. In the reversebinding mode, a hydrophilic aromatic ring can be positioned in the innercavity, allowing a carboxylate to hydrogen bond with Ser 117 and Ser117′. In the reverse binding mode a halogen-substituted hydrophobic ringcan be positioned in the outer cavity.

Diflunisal can reduce TTR acid-mediated amyloidogenesis. The structureof diflunisal (see Example 2) can be used as the basis for designing newcompounds that can inhibit TTR amyloidogenesis. See, for example,Verbeeck, R. K.; et al. Biochem. Pharm. 1980, 29, 571–576; andNuernberg, B.; Koehler, G.; Brune, K. Clin. Pharmacokin. 1991, 20,81–89.

The compound can have the formula:

where Ar¹ is an aryl or heteroaryl group, Ar¹ being optionallysubstituted with one or more of: halo, —R¹, —OR¹, —OC(═O)R¹, —OC(═O)OR¹,—OC(═O)NHR¹, —SR¹, —S(═O)R¹, —S(═O)₂R¹, —C(═O)R¹, —CO₂R¹, —C(═O)NHR¹,—NR¹R², —NHC(═O)R¹, NHC(═O)NHR¹, —NHC(═O)OR¹, or —NHS(═O)₂R¹.

Ar² is an aryl or heteroaryl group, Ar² being optionally substitutedwith one or more of: halo, —R¹, —OR¹, —OC(═O)R¹, —OC(═O)OR¹,—OC(═O)NHR¹, —SR¹, —S(═O)R¹, —S(═O)₂R¹, —C(═O)R¹, —CO₂R¹, —C(═O)NHR¹,—NR¹R², —NHC(═O)R¹, —NHC(═O)NHR¹, —NHC(═O)OR¹, or —NHS(═O)₂R¹.

Each R¹ is, independently, hydrogen, or a substituted or unsubstitutedalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl,heterocycloalkenyl, alkynyl, aryl, or heteroaryl group.

Each R² is, independently, hydrogen, or a substituted or unsubstitutedalkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl,heterocycloalkenyl, alkynyl, aryl, or heteroaryl group.

In certain circumstances, Ar¹ can be substituted or unsubstitutedphenyl. Ar² can independently be substituted or unsubstituted phenyl.Ar¹ and Ar² can simultaneously be substituted or unsubstituted phenyl.The substituents can be fluoro, chloro, hydroxy, —CO₂H, —CO₂Me, —OMe,—CH₂OH, or formyl. R¹ can be lower alkyl.

The compounds may be used in the form of pharmaceutically acceptablesalts derived from inorganic or organic acids and bases. Included amongsuch acid salts are the following: acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, the basic nitrogen-containinggroups can be quaternized with such agents as lower alkyl halides, suchas methyl, ethyl, propyl, and butyl chloride, bromides and iodides;dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamylsulfates, long chain halides such as decyl, lauryl, myristyl and stearylchlorides, bromides and iodides, aralkyl halides, such as benzyl andphenethyl bromides and others. Water or oil-soluble or dispersibleproducts are thereby obtained.

The compounds can stabilize TTR tetramers and inhibit formation of TTRamyloid. The compounds can be analogs of diflunisal characterized bysubtle structural changes. The compounds can be used to evaluatestructure-activity relationships as they pertain to TTR amyloidinhibition. Substitution patterns and the number of substituentsincluding halogens, carboxylates, acyl, alkoxy and hydroxyl can bevaried. Structure-activity data from other classes of compounds revealthat a carboxylate substituent or analogous anionic or H-bonding groupappears to be important, possibly participating in electrostaticinteractions with the M-ammonium groups of Lys 15 and 15′ or hydrogenbonding interactions with Ser 117 and 117′, while thehalogen-substituted hydrophobic ring compliments TTR's halogen bindingpockets. Both fluorine and chlorine-substituted aryls can be evaluated,including 2-fluoro-, 4-fluoro-, 3,5-difluoro-, 2,4-difluoro- and2,6-difluoro-. Iodine-substituted aryl groups may be less desirable dueto their lability and potential for acting as thyroxine agonists. Thecarboxylate (anionic) substituent can be absent in some analogs toevaluate its influence on fibril inhibition and plasma bindingselectivity. Compounds containing an aldehyde or alcohol functionalitycan be synthesized to evaluate the influence of a noncharged hydrogenbond acceptor or donor on binding selectivity and amyloid fibrilinhibition. The gem-diol form of the aldehyde can be the principlebinding species.

In general, the compounds can be synthesized by methods known in theart. One method of making the compounds is a Suzuki coupling:

-   -   BY₂=B(OH)₂, B(OR)₂, 9-BBN, B(CHCH₃CH(CH₃)₂)₂    -   X=I, Br, Cl, OSO₂(C_(n)F_(2n+1)), n=0, 1, 4    -   R₁=aryl, alkenyl, alkyl    -   R₂=aryl, alkenyl, benzyl, allyl, alkyl

For example, a biphenyl compound can be formed by a Suzuki coupling of aphenyl boronic acid with a bromobenzene or an iodobenzene. Appropriateprotecting groups may be needed to avoid forming side products duringthe preparation of a compound. For example, an amino substituent can beprotected by a suitable amino protecting group such as trifluoroacetylor tert-butoxycarbonyl. Other protecting groups and reaction conditionscan be found in T. W. Greene, Protective Groups in Organic Synthesis,(3rd, 1999, John Wiley & Sons, New York, N.Y.).

Pharmaceutical Compositions

The compounds described herein (e.g., diflunisal analogs,polychlorinated biphenyls, or benzoxazoles) may be formulated intopharmaceutical compositions that may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques.

The pharmaceutical compositions can include any of the compounds, orpharmaceutically acceptable derivatives thereof, together with anypharmaceutically acceptable carrier. The term “carrier” as used hereinincludes acceptable adjuvants and vehicles. Pharmaceutically acceptablecarriers that may be used in the pharmaceutical compositions of thisinvention include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as do natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions may be orally administered in any orallyacceptable dosage form including, but not limited to, capsules, tablets,aqueous suspensions or solutions. In the case of tablets for oral use,carriers which are commonly used include lactose and corn starch.Lubricating agents, such as magnesium stearate, are also typicallyadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried corn starch. When aqueous suspensions arerequired for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions may be administered inthe form of suppositories for rectal administration. These can beprepared by mixing the agent with a suitable non-irritating excipientwhich is solid at room temperature but liquid at the rectal temperatureand therefore will melt in the rectum to release the drug. Suchmaterials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions may also be administered topically,especially when the target of treatment includes areas or organs readilyaccessible by topical application, including diseases of the eye, theskin, or the lower intestinal tract. Suitable topical formulations arereadily prepared for each of these areas or organs.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds include, but are not limited to, mineraloil, liquid petrolatum, white petrolatum, propylene glycol,polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical compositions can be formulated in asuitable lotion or cream containing the active components suspended ordissolved in one or more pharmaceutically acceptable carriers. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions may also be administered by nasalaerosol or inhalation through the use of a nebulizer, a dry powderinhaler or a metered dose inhaler. Such compositions are preparedaccording to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated, and the particular mode of administration. It should beunderstood, however, that a specific dosage and treatment regimen forany particular patient will depend upon a variety of factors, includingthe activity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, rate of excretion,drug combination, and the judgment of the treating physician and theseverity of the particular disease being treated. The amount of activeingredient may also depend upon the therapeutic or prophylactic agent,if any, with which the ingredient is co-administered.

An effective amount of a pharmaceutical composition is the amount whichis required to confer a therapeutic effect on the treated patient, andwill depend on a variety of factors, such as the nature of theinhibitor, the size of the patient, the goal of the treatment, thenature of the pathology to be treated, the specific pharmaceuticalcomposition used, and the judgment of the treating physician. Forreference, see Freireich et al., Cancer Chemother. Rep. 1966, 50, 219and Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970,537. Dosage levels of between about 0.001 and about 100 mg/kg bodyweight per day, \ between about 0.1 and about 10 mg/kg body weight perday of the active ingredient compound may be useful.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Prevention of Transthyretin Amyloid Disease byChanging Protein Misfolding Energetics

Hundreds of human diseases, including the amyloidoses, are associatedwith protein misfolding. The 80 familial mutations that exacerbate [forexample, Val30→Met30 (V30M) and Leu55→Pro55 (L55P)] or ameliorate[Thr119→Met119 (T119M)] transthyretin (TTR) amyloid pathology providevaluable mechanistic insights. All disease-associated mutationscharacterized thus far destabilize the TTR tetramer, and many influencethe velocity of rate-limiting tetramer dissociation, with rapid ratesaccelerating and slow rates retarding amyloidosis. We took advantage ofthe mechanism by which T119M prevents disease in V30M compoundheterozygotes to develop small-molecule TTR amyloid inhibitors thatdramatically slowed the initial misfolding event (tetramer dissociation)required for partial monomer denaturation, enabling misassembly intoamyloid and other aggregates.

Hybrid tetramers were isolated to better understand the mechanism oftrans-suppression. Increasing T119M subunit stoichiometry relative toV30M [or L55P] shifted the maximum for acid-mediated fibril formation toa lower pH, decreased the overall yield of amyloid at physiologicallyaccessible pH's (>4.0), and slowed the rate of acid-induced (pH 4.4) andmethanol-mediated amyloidogenesis. Several small-molecule TTR amyloidfibril inhibitors have been discovered, a subset of which were studiedherein, including two drugs approved by the U.S. Food and DrugAdministration (FDA) (inhibitors 8 and 10) (Sacchettini et al., NatureRev. Drug Discovery 1, 267 (2002)). The influence of small-moleculeinhibitor binding on the yield and rate of wild-type (WT) TTR fibrilformation was similar to that of T119M subunit incorporation. However,the shift to a lower pH optimum for fibril formation was not observedwith all the inhibitors. These inhibitors function by binding to the twoequivalent thyroxine (T4) sites within the TTR tetramer, not themonomer.

Tetramer dissociation rates were measured by linking slow quaternarystructural changes to the unfolding transition, with a rate of 5×10⁵times that of dissociation (Hammarström et al., Proc. Natl. Acad. Sci.U.S.A. 99, 16427 (2002)). Denaturation-detected dissociation isirreversible because the concentration of urea used (>6.0 M) cannotsupport refolding. Increasing T119M subunit stoichiometry relative tothe V30M [or L55P] subunits revealed a dramatic TTR (1.8 μM) tetramerdissociation rate decrease (rate limiting for amyloidogenesis) in threedifferent denaturing environments (acidic pH, aqueous methanol, orurea), explaining the origin of disease prevention.

Measurements of the WT TTR tetramer (1.8 μM) dissociation rate in thepresence of inhibitors 6 through 10 (1.8 and 3.6 μM) showeddose-dependent slowing for all TTR-inhibitor complexes. The initial rateof tetramer dissociation was roughly inversely proportional to the molefraction of the tetramer bound to two inhibitors (T·I₂). In the case ofinhibitors 6, 7, and 9 (1.8 μM), the amplitude of the single exponentialcorrelated primarily with dissociation of the unliganded tetramer (and,to a lesser extent, T·I), implying that T·I₂ prevented tetramerdissociation in 6 M urea. In contrast, formation of T·I and T·I₂ forinhibitors 8 and 10 did not protect the tetramer substantially fromdissociation in urea, revealing that binding alone was insufficient. Theefficient inhibition observed in the case of 6, 7, and 9 (3.6 μM)resulted from the binding energy stabilizing the T·I₂ complex by freeenergies exceeding 2.3 kcal/mol (Delta G1=RT ln([T·I]/[T])=RTln([I]/Kd1) and Delta G2=RT ln([T·I₂]/[T])=RT ln{[I]2/(Kd1*Kd2)).Stabilizing T·I₂ relative to T by 2.7 kcal/mol would translate to atwo-order-of-magnitude decrease in the rate of TTR tetramerdissociation. The strong negatively cooperative binding of inhibitors 8and 10 (3.6 μM) dictates that binding to the second site (T·I₂, μMdissociation constants) would not further stabilize TTR relative tobinding to the first site (T·I). The nM dissociation constants (Kd1 andKd2) of inhibitors 6, 7, and 9 would ensure that ground-statestabilization (>2.3 kcal/mol) would be sufficient to substantiallyincrease the activation barrier for TTR tetramer dissociation, providedthat the inhibitors did not bind to and similarly stabilize thedissociative transition state. The inhibitor dissociation rates from theT·I₂ and T·I complex could also play a role in the efficacy ofinhibitors 6, 7, and 9. TTR saturated with inhibitor was immobilized byan antibody resin, over which aqueous buffer was passed at 5.0 ml/min toevaluate effective dissociation rates of 6 through 10. The bestinhibitors were those with the lowest apparent dissociation rates.

Although there is generally a very good correlation between theamyloidogenesis rates (acidic conditions) and tetramer dissociationrates (in urea) in the presence of inhibitors, this need not be thecase. Amyloidogenesis requires concentration-dependent misassembly afterdissociation. Thus, small molecules will generally be more effective atpreventing fibril formation than tetramer dissociation, especially whenthe inhibitor can keep the concentration of the monomeric amyloidogenicintermediate low (<3.6 μM), where fibril formation is very inefficient.Occasionally, tetramer dissociation rates measured in urea will notaccurately predict the rank ordering of inhibitor efficacy under acidicconditions. For example, the FDA-approved drug diflunisal (8) was a muchbetter amyloid inhibitor than a tetramer dissociation inhibitor. Alikely explanation for this observation is that Kd1 and/or Kd2 are lowerin acid than in urea (18). In addition, some inhibitors perform muchbetter under denaturing conditions than their binding constantsdetermined under physiological conditions would suggest. For example,compound 9 was more or equally efficient at preventing tetramerdissociation (urea) and fibril formation (acid) than was inhibitor 7,despite inhibitor 9 having Kd1 and Kd2 values that were 10 and 83 timesthat of 7, respectively (measured under physiological conditions). Thus,it is important to judge the efficacy of misfolding inhibitors under avariety of denaturing conditions and not just under physiologicalconditions.

Inclusion of T119M trans-suppressor subunits into tetramers otherwisecomposed of disease-associated subunits could decrease the rate oftetramer dissociation by stabilizing the tetrameric ground state to agreater extent than the transition state (as is the case with thesmall-molecule inhibitors) and/or by destabilizing the transition stateof dissociation. To distinguish between these possibilities, we comparedthe reconstitution kinetics of WT and T119M homotetramers. Refolding ofT119M monomers was rapid and within error of the folding rate of WT TTRmonomers. However, reassembly of T119M folded monomers was two orders ofmagnitude slower than the tetramerization of WT TTR monomers initiatedby urea dilution. The reassembly process is biphasic, which can beexplained by the presence of an observable intermediate in the assemblypathway (probably a dimer). In the opposite direction, the T119Mtetramer dissociates at 1/37 the rate exhibited by the WT TTR tetramer.These kinetic effects cannot be attributed to differences in tertiarystructural stability and/or tetramer stability. A direct comparison ofthe thermodynamic stability of WT and T119M monomers (employing anengineered monomeric TTR construct (M-TTR)) revealed a difference in thefree energy Delta Delta G for unfolding of only 0.4 kcal/mol, much lessthan the 2.1 and 2.7 kcal/mol required to explain the dissociation andassembly rate differences, respectively. A thermodynamic cycle analysisof T119M and WT TTR revealed that T119M prevents dissociation of thetetramer by destabilizing the dissociation transition state by approx3.1 kcal/mol, not by tetramer stabilization. According to this analysis,the T119M tetramer is actually destabilized by 0.9 kcal/mol relative toWT, further supporting a kinetic stabilization mechanism. Thefree-energy difference between WT and T119M tetramer dissociation cannotbe measured through urea-mediated unfolding because T119M denaturationin urea requires exceedingly long incubation periods (several weeks),during which TTR becomes modified. Comparisons of guanidinium chloride(GdmCl) and guanidinium thiocyanate (GdmSCN) denaturation curvesrevealed that WT TTR was more resistant to GdmCl denaturation than wasT119M, whereas the opposite was true in GdmSCN. These differences inmidpoints of denaturation can be attributed to differential anionstabilization, suggesting that the true thermodynamic stabilities ofthese proteins are very similar, although a quantitative analysis is notpossible in these chaotropes.

T119M trans-suppression is principally mediated by destabilization ofthe dissociative transition state, consistent with positioning of T119Mat the dimer-dimer interface. Increasing the dissociativetransition-state energy by 3.1 kcal/mol effectively prevents tetramerdissociation because the activation barrier becomes insurmountable(dissociation half-life t½ increases from approx 42 hours to >1500hours). Small-molecule binding similarly increases the activationbarrier associated with tetramer dissociation in a dose-dependentfashion, although this is mediated through tetramer stabilization(relative to transition state stabilization). The extent ofstabilization is maximal when the small-molecule dissociation constantsKd1 and Kd2 are as low as possible and the concentration of inhibitor isas high as possible. The concentrations used in our experiments forground-state stabilization are comparable to those observed in plasmafor numerous orally available drugs.

Small-molecule binding and trans-suppression increase the activationenergy associated with tetramer dissociation, the rate-limiting step ofTTR fibril formation. Establishing this analogy is important because itis known that trans-suppression prevents disease in V30M compoundheterozygotes. Kinetic stabilization of the native state is aparticularly attractive strategy, considering the emerging evidence thatsmall misfolded oligomers are neurotoxic. Discovering small-moleculebinders or developing a trans-suppression approach to tune the energylandscape of other pathologically relevant proteins with a predilectionto misfold should now be considered.

Example 2 Diflunisal Analogs Stabilize the Native State of Transthyretinand are Inhibitors of Transthyretin Amyloid Fibril Formation

Diflunisal (1) can reduce Transthyretin (TTR) amyloidogenesis. Forexample, under certain conditions (e.g., 3.6 TM TTR, 3.6 TM diflunisal,pH 4.4, 72 h, 37° C.), diflunisal reduces TTR amyloidogenesis by 63%.Under these conditions, doubling the diflunisal concentration (to 7.2TM) reduces amyloidogenesis by 97%. Diflunisal is one of the betteramyloid fibril inhibitors reported to date and orally administereddiflunisal is highly bioavailable, affording a sustained plasmaconcentration exceeding 100 TM at a dose of 250 mg twice daily. Becausediflunisal is a cyclooxygenase-2 inhibitor, long-term administrationcould lead to gastrointestinal side effects. Analogs of diflunisal thathave reduced or absent NSAID activity, but possess high affinity for TTRin blood plasma, are therefore optionally desirable. The structure ofdiflunisal can thus be used as the basis for designing new compoundsthat can inhibit TTR amyloidogenesis. See, for example, Verbeeck, R. K.;et al. Biochem. Pharm. 1980, 29, 571–576; and Nuernberg, B.; Koehler,G.; Brune, K. Clin. Pharmacokin. 1991, 20, 81–89.

Diflunisal analogs were synthesized using a Pd-mediated Suzuki couplingbetween an aryl halide and an aryl boronic acid. The synthesis ofanalogs 2–10 was achieved by acetylation of either 3- or 4-iodophenolwith acetic anhydride and pyridine, followed by Suzuki coupling with theappropriate fluorophenyl boronic acid under the standard Suzuki couplingreaction conditions, as shown in Scheme 1. Removal of the ester with Na⁰and MeOH (Zemplén conditions) provided phenols 2–10. See, for example,Miyaura, N.; Yanagi, T.; Suzuki, A. Synth. Commun. 1981, 11, 513–519;Sharp, M. J.; Snieckus, V. Tetrahedron Lett. 1985, 26, 5997–6000; Sharp,M. J.; Cheng, W.; Snieckus, V. Tetrahedron Lett. 1987, 28, 5093–5096;Pozsgay, V.; Nanasi, P.; Neszmelyi, A. Carbohydr. Res. 1981, 90,215–231; Jendralla, H.; Chen, L.-J. Synthesis 1990, 827–833; and Kelm,J.; Strauss, K. Spectrochim. Acta, Part A 1981, 37, 689–692, each ofwhich is incorporated by reference in its entirety. Acta, Part A 1981,37, 689–692, each of which is incorporated by reference in its entirety.

Diflunisal analog 11 was synthesized using solid-phase methods shown inScheme 2. 3-iodobenzoic acid was coupled to Wang resin via an esterlinkage, affording the resin-bound phenyliodide, which was then coupledto 2,4-difluorophenyl boronic acid, and cleaved from the resin with a1:1 mixture of TFA:CH₂Cl₂. See, for example, Guiles, J. W.; Johnson, S.G.; Murray, W. V. J. Org. Chem. 1996, 61, 5169–5171, which isincorporated by reference in its entirety.

Carboxylate-containing substrates 12–22 were assembled by coupling ofeither methyl-3-bromobenzoate or methyl-4-bromobenzoate (bothcommercially available) with the appropriate fluorophenyl boronic acidutilizing standard Suzuki coupling conditions (see above), as shown inScheme 3. The ester was then saponified with LiOH.H₂O to provide thecorresponding carboxylate. See, for example, Bumagin, N. A.; Bykov, V.V. Tetrahedron 1997, 53, 14437–14450; Ananthakrishnanadar, P.; Kannan,N. J. Chem. Soc., Perkin Trans. 2 1982, 1305–1308; Homsi, F.; Nozaki,K.; Hiyama, T. Tetrahedron Lett. 2000, 41, 5869–5872; and Hajduk, P. J.;et al. J. Med. Chem. 1997, 40, 3144–3150, each of which is incorporatedby reference in its entirety.

5-Iodosalicylic acid was esterified using TMS-CH₂N₂, and the phenol wasconverted into a methyl ether employing MeI. The protected salicylicacid was coupled with the various fluorophenyl boronic acids, andsubsequently deprotected by LiOH.H₂O saponification and BBr₃demethylation to provide salicylic acid derivatives 23–27, as shown inScheme 4. See, for example, Nicolaou, K. C.; et al. Chem. Eur. J. 1999,5, 2602–2621; and Chu-Moyer, M. Y.; et al. J. Med. Chem. 2002, 45,511–528.

3′,5′-Dihalo-4′-hydroxyl-containing analogs 28–31 were synthesized byfirst protecting the commercially available bromophenol as the methylether (MeI and K₂CO₃). Suzuki coupling with a (methoxycarbonylphenyl)boronic acid resulted in the formation of the fully protected biphenylsubstrates. BBr₃-mediated methyl ether cleavage and saponification withLiOH.H₂O provided the fully functionalized diflunisal analogs 28–31, asshown in Scheme 5.

Methyl ether and methyl ester analogs of diflunisal were synthesized byesterification of the carboxylic acid with TMS-diazomethane to provide32, optionally followed by etherification with MeI and K₂CO₃ and esterhydrolysis with LiOH.H₂O to afford 33. See Scheme 6.

A series of halogenated biphenyls 34–38 were assembled by Suzukicoupling of iodobenzene with a series of halogen-containing boronicacids, as shown in Scheme 7. See, for example, Patrick, T. B.;Willaredt, R. P. DeGonia, D. J. J. Org. Chem. 1985, 50, 2232–2235;Kuchar, M.; et al. Collection of Czechoslovak Chemical Communications1988, 53, 1862–1872; Allen, K. J.; Bolton, R.; Williams, G. H. J. Chem.Soc., Perkin Trans. 2 1983, 691–695; Nakada, M.; et al. Bull. Chem. Soc.Jpn. 1989, 62, 3122–3126; and Weingarten, H. J. Org. Chem. 1961, 26,730–733, each of which is incorporated by reference in its entirety.

Chlorinated biaryl aldehydes were assembled using3,5-dichloroiodobenzene and either 2-, 3- or 4-formylphenyl boronicacid, as shown in Scheme 8. Aldehydes 42–44, lacking the halogensubstitution, were prepared analogously. Aldehydes 39–41 were eitheroxidized with KMnO₄ in acetone/water to provide the correspondingcarboxylic acids 45–47 or reduced with NaBH₄ in MeOH to provide thecorresponding benzyl alcohols 48–50, Scheme 8. Reduction of thenon-chlorinated aldehydes 42–44 with NaBH₄ and MeOH produced thebiphenyl benzylic alcohols 51–53. See, for example, Song, X. P.; He, H.T.; Siahaan, T. J. Org. Lett. 2002, 4, 549–552; and Nicolaou, K. C.; etal. J. Am. Chem. Soc. 2001, 123, 9313–9323; Hashizume, H.; et al. Chem.Pharm. Bull. 1994, 42, 512–520; Indolese, A. F. Tetrahedron Lett. 1997,38, 3513–3516; Pridgen, L. N.; Snyder, L.; Prol, J. J. Org. Chem. 1989,54, 1523–1526; Huang, C. G.; Beveridge, K. A.; Wan, P. J. Am. Chem. Soc.1991, 113, 7676–7684; Wendeborn, S.; et al. Synlett. 1998, 6, 671–675;Stevens, C. V.; Peristeropoulou, M.; De Kimpe, N. Tetrahedron 2001, 57,7865–7870; Tanaka, K.; Kishigami, S.; Toda, F. J. Org. Chem. 1990, 55,2981–2983; and Clive, D. L. J.; Kang, S. Z. J. Org. Chem. 2001, 66,6083–6091, each of which is incorporated by reference in its entirety.

3′,5′-Difluoroformyl-functionalized biphenyls 54 and 55 were synthesizedvia Suzuki coupling of 3,5-difluorophenyl boronic acid with either 2- or3-iodobenzaldehyde, as shown in Scheme 9. All other inhibitors weresynthesized by similar methods and reported previously. Compounds 10,21, 35, 36 and 43 are commercially available.

Reagents and solvents were purchased from Aldrich, Lancaster, Acros,Combi-Blocks, Matrix and Pfaltz-Bauer. THF and CH₂Cl₂ were dried bypassage over Al₂O₃. Other solvents and reagents were obtained fromcommercial suppliers and were used without further purification unlessotherwise noted. Reactions were monitored by analytical thin layerchromatography (TLC) on silica gel 60 F₂₅₄ pre-coated plates withfluorescent indicator purchased from EM Science. Visualization of theTLC plates was accomplished by UV illumination, phosphomolybdic acidtreatment followed by heat or ceric ammonium molybdate treatmentfollowed by heat. Flash chromatography was performed using silica gel 60(230–400 mesh) from EM Science. The purity of new compounds that wereessential to the conclusions drawn in the text were determined by HPLC.Normal phase HPLC was performed with a Waters 600 pump/controller, aWaters 996 photodiode array detector and a Waters NovaPak silica column.The solvent system employed was hexanes and ethyl acetate, and gradientswere run from 50:50 hexanes:ethyl acetate to 0:100 hexanes:ethyl acetateover 30 min. Reverse phase HPLC was performed with a Waters 600pump/controller, a Waters 2487 dual wavelength detector and a Vydacprotein and peptide C18 column. Solvent system A was 95:5water:acetonitrile with 0.5% trifluoroacetic acid and solvent B was 5:95water:acetonitrile with 0.5% trifluoroacetic acid. Gradients were runfrom 100:0 A:B to 0:100 A:B over 20 min with a hold at 100% B for anadditional 10 min. Circular dichroism spectroscopy was performed on anAVIV Instruments spectrometer, model 202SF. NMR spectra were recorded ona Varian FT NMR spectrometer at a proton frequency of 400 MHz. Protonchemical shifts are reported in parts per million (ppm) with referenceto CHCl₃ as the internal chemical shift standard (7.26 ppm) unlessotherwise noted. Coupling constants are reported in hertz (Hz). Carbonchemical shifts are reported in parts per million (ppm) with referenceto CDCl₃ as the chemical shift standard (77.23 ppm) unless otherwisenoted. All mass spectra were obtained at The Scripps Research InstituteCenter for Mass Spectrometry or the University of Illinois MassSpectrometry Laboratory.

Compounds 2–10 were prepared according to Scheme 1. To a solution of theappropriate acetic acid-iodophenyl ester (1.0 equiv) dissolved in enoughtoluene to give a concentration of 0.05 M, was added a solution ofphenyl boronic acid (1.1 equiv) dissolved in EtOH to give a 0.6 Msolution with respect to the boronic acid. A 2 M aqueous solution ofNa₂CO₃ was added to give a final reaction concentration of 0.03 M withrespect to the acetic acid-iodophenyl ester, followed by addition ofPd(PPh₃)₄ (4.0 mol %). The reaction was heated to reflux under Ar for 20h, and upon completion, was cooled to rt and extracted with CH₂Cl₂ (2×),washed with brine (1×), dried over MgSO₄ and concentrated in vacuo. Theresidue was purified by flash chromatography (10:1 hexane:ethyl acetate)to afford the acetylated biphenyl.

A catalytic amount of Na⁰ was added to a solution of the acetylatedbiphenyl in MeOH to provide a final reaction concentration of 0.3 M. Thereaction was allowed to stir at rt under Ar for 12 h, after which Dowex50W-X8 cation exchange resin was added to neutralize the reactionmixture. The resin was filtered and the filtrate was concentrated invacuo and flash chromatographed (3:1 hexane:ethyl acetate) to afford thehydroxybiphenyl products as white solids in 22–75% yields.

2′,4′-Difluorobiphenyl-3-ol (2). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.63 (br s,1H), 7.54 (td, 1H, J=8.9, 6.7 Hz), 7.34 (ddd, 1H, J=11.1, 9.2, 2.6 Hz),7.27 (m, 1H), 7.17 (tdd, 1H, J=8.3, 2.6, 1.2 Hz), 6.92 (m, 2H), 6.81(ddd, 1H, J=8.1, 2.5, 1.0 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ 162.8,160.3, 157.4, 135.4, 131.8, 129.7, 119.4, 115.6, 114.9, 111.9, 104.4.HRESIMS calculated for C₁₂H₈F₂O (M−H) 205.0466, found 205.0465. Normalphase HPLC retention time: 10.5 min. Reverse phase HPLC retention time:1.3 min. >99% pure.

2′,4′-Difluorobiphenyl-4-ol (3). ¹H NMR (DMSO-d₆, 400 MHz) δ 7.49 (td,1H, J=9.4, 8.6 Hz), 7.34 (AA′XX′, 2H, J_(AA′)=J_(XX′)=2.5 Hz, J_(XA)=8.7Hz, J_(X′A′)=8.5 Hz, J_(X′A)=0.3 Hz, J_(XA′)=0.3 Hz, ν_(A)=ν_(A′)=2934.1Hz, ν_(X)=ν_(X′)=2746.2 Hz), 7.28 (ddd, 2H, J=11.3, 9.4, 2.6 Hz), 7.13(dddd, 1H, J=8.3, 7.5, 2.8, 1.0 Hz), 6.87 (AA′XX′, 2H, as above). ¹³CNMR (DMSO-d₆, 100 MHz) δ 162.3, 160.0, 157.2, 131.4, 129.9, 124.8,115.4, 111.8, 104.3. HRESIMS calculated for C₁₂H₈F₂O (M−H) 205.0464,found 205.0465. Normal phase HPLC retention time: 11.2 min. Reversephase HPLC retention time: 12.6 min. >98% pure.

3′,5′-Difluorobiphenyl-3-ol (8). ¹H NMR (DMSO-d₆, 400 MHz) δ 9.65 (br s,1H), 7.34 (m, 2H), 7.28 (t, 1H, J=7.9 Hz), 7.19 (tt, 1H, J=9.1, 2.2 Hz),7.13 (ddd, 1H, J=7.8, 1.8, 1.0 Hz), 7.08 (t, 1H, J=2.1 Hz), 6.86 (ddd,1H, J=8.0, 2.4, 1.0 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ 162.9, 158.0,144.1, 139.1, 130.1, 117.6, 115.7, 109.7, 102.6. HRESIMS calculated forC₁₂H₈F₂O (M−H) 205.0465, found 205.0468. Normal phase HPLC retentiontime: 11.4 min. Reverse phase HPLC retention time: 12.9 min. >99% pure.

3′,5′-Difluorobiphenyl-4-ol (9). ¹H NMR (CDCl₃, 400 MHz) δ 7.44 (AA′XX′,2H, J_(AA′)=J_(XX′)=3.0 Hz, J_(XA)=8.0 Hz, J_(X′A′)=8.5 Hz, J_(X′A)=0.7Hz, J_(XA′)=0.5 Hz, ν_(A)=ν_(A′)=2973.8 Hz, ν_(X)=ν_(X′)=2766.0 Hz),7.05 (dtd, 2H, J=6.6, 2.4, 0.7 Hz), 6.92 (AA′XX′, 2H, as above), 6.74(tt, 1H, J=8.9, 2.4 Hz), 5.11 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 164.7,156.1, 144.2, 131.8, 128.6, 116.1, 109.6, 102.1. HRESIMS calculated forC₁₃H₈Cl₂O₂ (M−H) 205.0465, found 205.0465. Normal phase HPLC retentiontime: 10.8 min. Reverse phase HPLC retention time: 12.9 min. >99% pure.

2′,4′-Difluorobiphenyl-3-carboxylic acid (11). Compound 11 was preparedaccording to Scheme 2. 3-Iodobenzoic acid (200 mg, 0.81 mmol), DIEA (140TL, 0.81 mmol), EDCI.HCl and HOBt were added to a solution of Wang resin(265 mg, 0.67 mmol, 2.53 mmol/g) swelled in CH₂Cl₂ (10 mL). Afterrigorous shaking on a peptide shaker for 22 h at rt, the solvent wasremoved and the resin was washed with DMF (3×10 mL) and CH₂Cl₂ (3×10 mL)and dried thoroughly in vacuo.

2,4-Difluorophenyl boronic acid (112 mg, 0.71 mmol), K₂CO₃ (98 mg, 0.71mmol) and Pd₂(dba)₃ (4 mg, 0.01 mmol) were added to a solution offunctionalized Wang resin (140 mg, 0.35 mmol) swelled in DMF (2 mL).After stirring at rt, the reaction was filtered and the resin was washedwith DMF (3×), H₂O (3×), CH₂Cl₂ (3×) and MeOH (3×) and dried thoroughlyin vacuo.

A solution of TFA:CH₂Cl₂ (3 mL 1:1) was added to functionalized resin(140 mg, 0.35 mmol) and shaken vigorously on a peptide shaker for 13 hat rt. After completion, the reaction was filtered, the resin was washedwith CH₂Cl₂ (3×), the filtrate was concentrated in vacuo and purified byflash chromatography (2:1 hexane:ethyl acetate, 0.5% acetic acid) toafford 11 (81 mg, 100%) as a white solid. ¹H NMR (DMSO-d₆, 400 MHz) δ13.19 (br s, 1H), 8.07 (q, 1H, J=1.7 Hz), 7.99 (dt, 1H, J=7.9, 1.6 Hz),7.78 (dq, 1H, J=7.8, 1.3 Hz), 7.64 (m, 2H), 7.40 (ddd, 1H, J=11.1, 8.8,2.5 Hz), 7.22 (tdd, 1H, J=8.4, 2.8, 1.0 Hz). ¹³C NMR (DMSO-d₆, 100 MHz)δ 167.0, 160.7, 160.4, 134.5, 133.0, 132.0, 131.3, 129.4, 129.1, 128.7,123.9, 112.2, 104.6. HRESIMS calculated for C₁₃H₈F₂O₂ (M−H) 233.0414,found 233.0426. Normal phase HPLC retention time: 13.7 min. Reversephase HPLC retention time: 12.5 min. >99% pure.

Compounds 12–22 were prepared according to Scheme 3. To a solution ofthe appropriate methyl bromobenzoate (1.0 equiv) dissolved in enoughtoluene to give a concentration of 0.1 M, was added a solution of phenylboronic acid (2.0 equiv) dissolved in EtOH to give a 1.0 M solution ofboronic acid. A 2 M aqueous solution of Na₂CO₃ was added to give a finalreaction concentration of 0.06 M with respect to the bromobenzoate,followed by addition of Pd(PPh₃)₄ (10.0 mol %). The reaction was stirredat 70° C. under Ar for 25 h, and upon completion, was cooled to rt andextracted with CH₂Cl₂ (2×), washed with brine (1×), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(10:1 hexane:ethyl acetate) to afford the methyl ester.

To a solution of methyl ester (1.0 equiv) in THF:MeOH:H₂O (1:1:1) at aconcentration of 0.06 M, was added LiOH.H₂O (3.0 equiv). The reactionwas stirred at rt for 4 h, and upon completion, was acidified with 30%HCl, extracted with ethyl acetate (3×5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(CH₂Cl₂, 1% MeOH, 0.2% acetic acid) to afford the biphenyl carboxylicacids as white solids in 6–93% yields.

2′,4′-Difluorobiphenyl-4-carboxylic acid (12). ¹H NMR (DMSO-d₆, 400 MHz)δ 13.09 (br s, 1H), 8.04 (AA′XX′, 2H, J_(AA′)=J_(XX′)=2.0 Hz,J_(XA)=J_(X′A′)=8.0 Hz, J_(X′A)=J_(XA′)=0.7 Hz, ν_(A)=ν_(A′)=3213.3 Hz,ν_(X)=ν_(X′)=3056.2 Hz), 7.65 (AA′XX′, 2H, as above), 7.63 (m, 1H), 7.38(ddd, 1H, J=11.2, 9.0, 2.8 Hz), 7.21 (td, 1H, J=8.4, 2.2 Hz). ¹³C NMR(DMSO-d₆, 100 MHz) δ 167.1, 160.8, 158.0, 138.6, 132.1, 130.1, 129.6,129.0, 123.9, 112.2, 104.7. HRESIMS calculated for C₁₃H₈F₂O₂ (M−H)233.0414, found 233.0407. Normal phase HPLC retention time: 13.3 min.Reverse phase HPLC retention time: 12.6 min. >99% pure.

2′-Fluorobiphenyl-3-carboxylic acid (15). ¹H NMR (CD₃OD, 400 MHz) δ 8.18(q, 1H, J=1.4 Hz), 8.03 (dt, 1H, J=7.8, 1.3 Hz), 7.76 (dq, 1H, J=7.7,1.5 Hz), 7.55 (t, 1H, J=7.8 Hz), 7.48 (td, 1H, J=7.8, 1.7 Hz), 7.38(dddd, 1H, J=8.3, 7.5, 5.1, 1.8 Hz), 7.26 (td, 1H, J=7.6, 1.3 Hz), 7.20(ddd, 1H, J=11.0, 8.2, 1.2 Hz). ¹³C NMR (CD₃OD, 100 MHz) δ 169.7, 161.2,137.5, 134.6, 132.4, 132.0, 131.3, 130.1, 129.9, 129.5, 126.0, 117.2.HRESIMS calculated for C₁₃H₉FO₂ (M−H) 215.0508, found 215.0498. Normalphase HPLC retention time: 10.6 min. Reverse phase HPLC retention time:12.1 min. >99% pure.

2′-Fluorobiphenyl-4-carboxylic acid (16). ¹H NMR (DMSO-d₆, 400 MHz) δ13.10 (br s, 1H), 8.05 (AA′XX′, 2H, J_(AA′)=J_(XX′)=1.7 Hz,J_(XA)=J_(X′A′)=8.5 Hz, J_(X′A=J) _(XA′)=0.3 Hz, ν_(A)=ν_(A′)=3217.9 Hz,ν_(X)=ν_(X′)=3070.0 Hz), 7.67 (AA′XX′, 2H, as above), 7.58 (td, 1H,J=8.0, 1.8 Hz), 7.34 (m, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 167.1, 159.1,139.4, 130.8, 130.3, 130.2, 129.6, 129.0, 127.3, 125.1, 116.2. HRESIMScalculated for C₁₃H₉FO₂ (M−H) 215.0508, found 215.0515. Normal phaseHPLC retention time: 12.3 min. Reverse phase HPLC retention time: 12.2min. >99% pure.

3′,5′-Difluorobiphenyl-3-carboxylic acid (17). ¹H NMR (acetone-d₆, 400MHz) δ 8.30 (td, 1H, J=2, 0.5 Hz), 8.10 (dtd, 1H, J=7.6, 1.1, 0.5 Hz),7.97 (ddd, 1H, J=7.8, 2.0, 1.1 Hz), 7.64 (td, 1H, J=7.8, 0.6 Hz), 7.39(m, 2H), 7.06 (tt, 1H, J=9.3, 2.4 Hz). ¹³C NMR (acetone-d₆, 100 MHz) δ167.4, 165.6, 163.2, 144.6, 139.8, 132.5, 132.4, 130.6, 130.3, 128.9,111.0, 103.7. HRESIMS calculated for C₁₃H₈F₂O₂ (M−H) 233.0414, found233.0425. Normal phase HPLC retention time: 13.5 min. Reverse phase HPLCretention time: 12.7 min. >99% pure.

3′,5′-Difluorobiphenyl-4-carboxylic acid (18). ¹H NMR (DMSO-d₆, 400 MHz)δ 13.15 (br s, 1H), 8.02 (d, 2H, J=8.2 Hz), 7.85 (d, 2H, J=8.5 Hz), 7.49(m, 2H), 7.26 (tt, 1H, J=9.4, 2.4 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ166.4, 164.1, 161.7, 142.6, 141.6, 130.9, 130.0, 127.1, 110.2, 103.5.HRESIMS calculated for C₁₃H₈F₂O₂ (M−H) 233.0414, found 233.0423. Normalphase HPLC retention time: 13.0 min. Reverse phase HPLC retention time:12.8 min. >99% pure.

2′,6′-Difluorobiphenyl-3-carboxylic acid (19). ¹H NMR (DMSO-d₆, 400 MHz)δ 8.03 (dt, 1H, J=7.8, 1.6 Hz), 8.00 (m, 1H), 7.72 (dt, 1H, J=7.8, 1.4Hz), 7.64 (t, 1H, J=7.7 Hz), 7.53 (m, 1H), 7.26 (t, 2H, J=8.3 Hz). ¹³CNMR (DMSO-d₆, 100 MHz) δ 167.7, 158.7, 135.0, 132.2, 131.4, 131.1,129.9, 129.5, 129.5, 112.8, 110.9. HRESIMS calculated for C₁₃H₈F₂O₂(M−H) 233.0414, found 233.0410. Normal phase HPLC retention time: 12.1min. Reverse phase HPLC retention time: 12.1 min. >97% pure.

2′,6′-Difluorobiphenyl-4-carboxylic acid (20). ¹H NMR (DMSO-d₆, 400 MHz)δ 8.06 (AA′XX′, 2H, J_(AA′)=J_(XX′)=2.0 Hz, J_(XA)=J_(X′A′)=8.0 Hz,J_(X′A)=J_(XA′)=0.7 Hz, ν_(A)=ν_(A′)=3243.6 Hz, ν_(X)=ν_(X′)=3018.6 Hz),7.60 (AA′XX′, 2H, as above), 7.54 (m, 1H), 7.27 (t, 2H, J=8.3 Hz). ¹³CNMR (DMSO-d₆, 100 MHz) δ 171.0, 164.0, 134.1, 125.7, 122.0, 121.9,121.1, 103.4. HRESIMS calculated for C₁₃H₈F₂O₂ (M−H) 233.0414, found233.0425. Normal phase HPLC retention time: 14.5 min. Reverse phase HPLCretention time: 12.1 min. >99% pure.

Biphenyl-4-carboxylic acid (22). ¹H NMR (DMSO-d₆, 400 MHz) δ 13.07 (brs, 1H), 8.03 (AA′XX′, 2H, J_(AA′)=J_(XX′)=1.8 Hz, J_(XA)=J_(X′A′)=8.3Hz, J_(X′A)=j_(XA′)=0.3 Hz, ν_(A)=ν_(A′)=3210.7 Hz, ν_(X)=ν_(X′)=3122.0Hz), 7.81 (AA′XX′, 2H, as above), 7.75 (m, 2H), 7.51 (tt, 2H, J=7.2, 1.1Hz), 7.43 (tt, 1H, J=7.4, 1.2 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ 167.2,144.2, 139.0, 130.0, 129.8, 129.1, 128.3, 127.0, 126.8. HREIMScalculated for C₁₃H₁₀O₂ (M+) 198.0683, found 198.0683. Normal phase HPLCretention time: 13.8 min. Reverse phase HPLC retention time: 12.2min. >99% pure.

Methyl-5-iodo-2-methoxybenzoate. TMS-diazomethane (19.25 mL, 38.50 mmol,2 M solution in hexane) was added to a solution of 5-iodosalicylic acid(5.08 g, 19.24 mmol) in MeOH (20 mL) and stirred at rt for 11 h. Uponcompletion, the reaction was concentrated in vacuo and the residue wascarried onto the next step without further purification.

Methyl iodide (2.40 mL, 38.48 mmol) and K₂CO₃ (10.60 g, 76.96 mmol) wereadded to a solution of 5-iodo-2-methoxybenzoate (5.37 g, 19.24 mmol) inDMF (20 mL) and stirred at rt under Ar for 24 h. Upon completion, ethylacetate was added and the reaction was washed with 1% HCl (2×20 mL),brine (1×), dried over MgSO₄ and concentrated in vacuo. The residue waspurified by flash chromatography (3:1 hexane:ethyl acetate) to affordmethyl-5-iodo-2-methoxybenzoate (4.93 g, 88%) as a white solid. See, forexample, Corey, E. J.; Myers, A. G. J. Am. Chem. Soc. 1985, 107,5574–5576, which is incorporated by reference in its entirety. ¹H NMR(DMSO-d₆, 400 MHz) δ 7.90 (d, 1H, J=2.4 Hz), 7.80 (dd, 1H, J=8.8, 2.4Hz), 6.96 (d, 1H, J=9.0 Hz), 3.81 (s, 3H), 3.79 (s, 3H). ¹³C NMR(DMSO-d₆, 100 MHz) δ 164.7, 158.0, 141.6, 138.5, 122.2, 115.2, 82.1,55.9, 52.0. HREIMS calculated for C₉H₉IO₃ (M+) 291.9608, found 291.9596.

Compounds 23–27 were prepared according to Scheme 4. To a solution ofmethyl-5-iodo-2-methoxybenzoate (1.0 equiv) dissolved in enough tolueneto give a concentration of 0.08 M, was added a solution of phenylboronic acid (2.0 equiv) dissolved in EtOH to give a 0.8 M solution ofboronic acid. A 2 M aqueous solution of Na₂CO₃ was added to give a finalreaction concentration of 0.06 M with respect to the methoxybenzoate,followed by addition of Pd(PPh₃)₄ (10.0 mol %). The reaction was stirredat 60° C. under Ar for 15 h, and upon completion, was cooled to rt andextracted with CH₂Cl₂ (2×), washed with brine (1×), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(3:1 hexane:ethyl acetate) to afford the methylated salicylates.

To a solution of the methylated salicylate (1.0 equiv) in enough CH₂Cl₂to give a concentration of 0.06 M, was added BBr₃ (2.0 equiv, 1 Msolution in CH₂Cl₂). The reaction was stirred at rt under Ar for 4 h,and upon completion, was quenched with H₂O (10 mL), extracted withCH₂Cl₂ (2×), washed with brine (1×), dried over MgSO₄ and concentratedin vacuo. The residue was carried onto the next step without furtherpurification.

To a solution of methyl ester (1.0 equiv) in THF:MeOH:H₂O (1:1:1) at aconcentration of 0.06 M, was added LiOH.H₂O (3.0 equiv). The reactionwas stirred at rt for 4 h, and upon completion, was acidified with 30%HCl, extracted with ethyl acetate (3×5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(CH₂Cl₂, 1% MeOH, 0.2% acetic acid) to afford the biphenyl salicylatesas white solids in 12–42% yields.

4′-Fluoro-4-hydroxybiphenyl-3-carboxylic acid (23). ¹H NMR (CD₃OD, 400MHz) δ 8.01 (d, 1H, J=2.5 Hz), 7.65 (dd, 1H, J=8.7, 2.5 Hz), 7.51 (m,2H), 7.11 (tt, 2H, J=10.0, 3.0 Hz), 6.97 (d, 1H, J=8.7 Hz. ¹³C NMR(CD₃OD, 100 MHz) δ 173.5, 165.0, 162.7, 137.7, 135.1, 132.6, 129.6,129.3, 118.9, 116.7, 116.6, 114.2. HRESIMS calculated for C₁₃H₉FO₃ (M−H)231.0459, found 231.0457. Normal phase HPLC retention time: 14.2 min.Reverse phase HPLC retention time: 12.8 min. >99% pure.

2′-Fluoro-4-hydroxybiphenyl-3-carboxylic acid (24). ¹H NMR (CD₃OD, 400MHz) δ 7.98 (dd, 1H, J=2.2, 1.4 Hz), 7.59 (ddd, 1H, J=8.7, 2.4, 1.7 Hz),7.36 (td, 1H, J=7.8, 1.7 Hz), 7.26 (dddd, 1H, J=9.9, 7.4, 4.9, 1.7 Hz),7.16 (td, 1H, J=7.5, 1.2 Hz), 7.10 (ddd, 1H, J=11.1, 8.2, 1.3 Hz), 6.95(d, 1H, J=8.5 Hz). ¹³C NMR (CD₃OD, 100 MHz) δ 173.5, 162.9, 162.4,137.2, 131.8, 130.2, 130.1, 129.1, 128.1, 125.8, 118.5, 117.1, 114.0.HRESIMS calculated for C₁₃H₉FO₃ (M−H) 231.0457, found 231.0446. Normalphase HPLC retention time: 13.8 min. Reverse phase HPLC retention time:12.7 min. >99% pure.

3′,5′-Difluoro-4-hydroxybiphenyl-3-carboxylic acid (25). ¹H NMR (CD₃OD,400 MHz) δ 8.07 (d, 1H, J=2.5 Hz), 7.73 (dd, 1H, J=8.5, 2.7 Hz), 7.15(m, 2H), 7.01 (d, 1H, J=8.9 Hz), 6.86 (tt, 1H, J=9.0, 2.5 Hz). ¹³C NMR(CD₃OD, 100 MHz) δ 173.3, 166.3, 163.8, 145.1, 135.2, 131.0, 129.8,119.2, 114.4, 110.4, 103.0. HRESIMS calculated for C₁₃H₈F₂O₃ (M−H)249.0363, found 249.0356. Normal phase HPLC retention time: 14.5 min.Reverse phase HPLC retention time: 13.3 min. >99% pure.

2′,4′-Dichloro-4-hydroxybiphenyl-3-carboxylic acid (26). ¹H NMR (CD₃OD,400 MHz) δ 7.83 (d, 1H, J=2.2 Hz), 7.70 (d, 1H, J=2.0 Hz), 7.58 (dd, 1H,J=8.6, 2.4 Hz), 7.48 (ABX, 1H, J_(AB)=8.4 Hz, J_(AX)=2.2 Hz, J_(BX)=0.0Hz, ν_(A)=2989.4 Hz, ν_(B)=2973.0 Hz), 7.44 (ABX, 1H, as above), 7.06(d, 1H, J=8.7 Hz). ¹³C NMR (CD₃OD, 100 MHz) δ 171.6, 160.8, 137.5,136.4, 132.8, 132.6, 132.4, 130.8, 129.2, 128.5, 127.7, 117.2, 112.9.HRESIMS calculated for C₁₃H₈Cl₂O₃ (M−H) 280.9772, found 280.9782. Normalphase HPLC retention time: 13.1 min. Reverse phase HPLC retention time:14.4 min. >99% pure.

4-Hydroxybiphenyl-3-carboxylic acid (27). ¹H NMR (CD₃OD, 400 MHz) δ 8.08(d, 1H, J=2.4 Hz), 7.73 (dd, 1H, J=8.7, 2.3 Hz), 7.54 (m, 2H), 7.41 (tt,2H, J=7.3, 1.8 Hz), 7.29 (tt, 1H, J=7.8, 1.7 Hz), 7.38 (dddd, 1H, J=8.8,6.4 Hz), 7.05 (d, 1H, J=8.7 Hz), 6.93 (m, 1H), 6.90 (ddd, 1H, J=7.3, 1.9Hz), 7.00 (d, 1H, J=8.5 Hz). ¹³C NMR (CD₃OD, 100 MHz) δ 161.5, 140.1,134.0, 132.4, 128.7, 128.3, 126.9, 126.3, 117.5, 112.9. HRESIMScalculated for C₁₃H₁₀O₃ (M−H) 213.0552, found 213.0545. Normal phaseHPLC retention time: 12.9 min. Reverse phase HPLC retention time: 12.6min. >99% pure.

4-Bromo-2,6-difluoroanisole. Methyl iodide (580 TL, 10.06 mmol) andK₂CO₃ (2.80 g, 20.12 mmol) were added to a solution of4-bromo-2,6-difluorophenol (1.05 g, 5.03 mmol) in DMF (10 mL) andstirred at rt under Ar for 24 h. Upon completion, ethyl acetate wasadded and the reaction was washed with 1% HCl (2×20 mL), brine (1×),dried over MgSO₄ and concentrated in vacuo. The residue was purified byflash chromatography (hexane) to afford 4-bromo-2,6-difluoroanisole (747mg, 67%) as a white solid. See, for example, Chambers, R. D.; et al. J.Fluorine Chem. 2000, 102, 169–174, which is incorporated by reference inits entirety. ¹H NMR (CDCl₃, 400 MHz) δ 7.06 (m, 2H), 3.97 (q, 3H, J=1.1Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 155.8, 136.3, 116.2, 113.8, 61.9. LREIMSfound for C₇H₅F₂OBr (M+) 223.0.

4-Bromo-2,6-dichloroanisole. Methyl iodide (467 TL, 8.12 mmol) and K₂CO₃(2.24 g, 16.24 mmol) were added to a solution of4-bromo-2,6-dichlorophenol (982 mg, 4.06 mmol) in DMF (10 mL) andstirred at rt under Ar for 40 min. Upon completion, ethyl acetate wasadded and the reaction was washed with 1% HCl (2×20 mL), brine (1×),dried over MgSO₄ and concentrated in vacuo. The residue was purified byflash chromatography (hexane) to afford 4-bromo-2,6-dichloroanisole (768mg, 74%) as a white solid. See, for example, Li, J.; et al. J. Med.Chem. 1996, 39, 1846–1856, which is incorporated by reference in itsentirety. ¹H NMR (DMSO-d₆, 400 MHz) δ 7.75 (s, 2H), 3.81 (s, 3H). ¹³CNMR (DMSO-d₆, 100 MHz) δ 151.3, 131.5, 129.6, 116.5, 60.6. HREIMScalculated for C₇H₅BrCl₂O (M+) 253.8905, found 253.8901.

Compounds 28–31 were prepared according to Scheme 5. To a solution ofthe appropriate halo-anisole (1.0 equiv) dissolved in enough toluene togive a concentration of 0.25 M, was added a solution of phenyl boronicacid (2.0 equiv) dissolved in EtOH to give a 1.5 M solution of boronicacid. A 2 M aqueous solution of Na₂CO₃ was added to give a finalreaction concentration of 0.08 M with respect to the halo-anisole,followed by addition of Pd(PPh₃)₄ (10.0 mol %). The reaction was stirredat 65° C. for 17 h, and upon completion, was cooled to rt and extractedwith CH₂Cl₂ (2×), washed with brine (1×), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(20:1 hexane:ethyl acetate) to afford the methylated biphenyl as a whitesolid.

To a solution of the methylated biphenyl (1.0 equiv) in enough CH₂Cl₂ togive a concentration of 0.20 M, was added BBr₃ (2.0 equiv, 1M solutionin CH₂Cl₂). The reaction was stirred at rt under Ar for 3 h, and uponcompletion, was quenched with H₂O (10 mL), extracted with CH₂Cl₂ (2×),washed with brine (1×), dried over MgSO₄ and concentrated in vacuo. Theresidue was carried onto the next step without further purification.

To a solution of methyl ester (1.0 equiv) in THF:MeOH:H₂O (1:1:1) at aconcentration of 0.04 M, was added LiOH.H₂O (3.0 equiv). The reactionwas stirred at rt for 5 h, and upon completion, was acidified with 30%HCl, extracted with ethyl acetate (3×5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(CH₂Cl₂, 1% MeOH, 0.2% acetic acid) to afford the biphenyl products aswhite solids in 14–39% yields.

3′,5′-Difluoro-4′-hydroxybiphenyl-3-carboxylic acid (28). ¹H NMR(DMSO-d₆, 400 MHz) δ 10.60 (br s, 1H), 8.14 (t, 1H, J=1.7 Hz), 7.91 (dt,1H, J=7.7, 1.1 Hz), 7.88 (ddd, 1H, J=8.0, 2.0, 1.1 Hz), 7.55 (t, 1H,J=7.9 Hz), 7.41 (m, 2H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 167.3 154.0,151.5, 138.4, 133.6, 131.6, 130.8, 129.9, 129.4, 128.4, 127.1, 110.3.HRESIMS calculated for C₁₃H₈F₂O₃ (M−H) 249.0363, found 249.0358. Normalphase HPLC retention time: 18.3 min. Reverse phase HPLC retention time:10.5 min. >98% pure.

3′,5′-Difluoro-4′-hydroxybiphenyl-4-carboxylic acid (29). ¹H NMR(DMSO-d₆, 400 MHz) δ 7.98 (AA′XX′, 2H, J_(AA′)=J_(XX′)=1.7 Hz,J_(XA)=J_(X′A′)=8.2 Hz, j_(X′A)=J_(XA′)=0.5 Hz, ν_(A)=ν_(A′)=3189.9 Hz,ν_(X)=ν_(X′)=3122.0 Hz), 7.81 (AA′XX′, 2H, as above), 7.51 (m, 2H). ¹³CNMR (DMSO-d₆, 100 MHz) δ 167.7, 154.5, 142.5, 136.0, 130.5, 130.5,130.4, 126.9, 111.0. HRESIMS calculated for C₁₃H₈F₂O₃ (M−H). 249.0363,found 249.0375. Normal phase HPLC retention time: 18.9 min. Reversephase HPLC retention time: 10.2 min. >99% pure.

3′,5′-Dichloro-4′-hydroxybiphenyl-3-carboxylic acid (30). ¹H NMR(DMSO-d₆, 400 MHz) δ 8.13 (t, 1H, J=1.6 Hz), 7.91 (m, 2H), 7.70 (s, 2H),7.56 (t, 1H, J=7.8 Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ 167.2, 149.0,137.9, 132.2, 131.6, 130.8, 129.3, 128.4, 127.1, 126.8, 122.9, 123.0.HRESIMS calculated for C₁₃H₈Cl₂O₃ (M−H) 280.9772, found 280.9767. Normalphase HPLC retention time: 16.2 min. Reverse phase HPLC retention time:11.6 min. >99% pure.

3′,5′-Dichloro-4′-hydroxybiphenyl-4-carboxylic acid (31). ¹H NMR(DMSO-d₆, 400 MHz) δ 7.98 (AA′XX′, 2H, J_(AA′)=J_(XX′)=1.7 Hz,J_(XA)=J_(X′A′)=8.1 Hz, j_(′=J) _(XA′)=0.5 Hz, ν_(A)=ν_(A′)=3189.9 Hz,ν_(X)=ν_(X′)=3110.0 Hz), 7.81 (AA′XX′, 2H,as above), 7.78 (s, 2H). ¹³CNMR (DMSO-d₆, 100 MHz) δ 167.2, 141.8, 141.7, 134.7, 129.9, 129.7,126.9, 126.4, 123.0. HRESIMS calculated for C₁₃H₈Cl₂O₃ (M−H) 280.9772,found 280.9785. Normal phase HPLC retention time: 15.9 min. Reversephase HPLC retention time: 11.4 min. >97% pure.

Methyl-2′,4′-difluoro-4-hydroxybiphenyl-3-carboxylate (32). Compounds 32and 33 were prepared according to Scheme 6. TMS-diazomethane (5.87 mL,11.75 mmol, 2 M solution in hexane) was added to a solution ofdiflunisal (1.03 g, 4.11 mmol) in MeOH (10 mL) and stirred at rt for 5h. Upon completion, the reaction was concentrated in vacuo and theresidue was purified by flash chromatography (10:1 hexane:ethyl acetate)to afford 32 (774 mg, 71%) as a white solid. ¹H NMR (CDCl₃, 400 MHz) δ7.97 (dd, 1H, J=2.2, 1.3 Hz), 7.59 (dt, 1H, J=8.8, 2.1 Hz), 7.36 (dq,1H, J=7.7, 1.5 Hz), 7.48 (td, 1H, J=7.8, 1.7 Hz), 7.38 (dddd, 1H, J=8.8,6.4 Hz), 7.05 (d, 1H, J=8.7 Hz), 6.93 (m, 1H), 6.90 (ddd, 1H, J=10.6,8.9, 2.5 Hz), 3.96 (s, 3H). ¹³C NMR (CDCl₃, 100 MHz) δ 170.6, 163.6,161.3, 158.5, 136.4, 131.2, 130.3, 126.2, 124.2, 118.0, 112.6, 111.8,104.6, 52.6, 124.2. HRFABMS calculated for C₁₄H₁₀F₂O₃ (M+) 264.0596,found 264.0598. Normal phase HPLC retention time: 6.9 min. Reverse phaseHPLC retention time: 14.7 min. >99% pure.

2′,4′-Difluoro-4-methoxybiphenyl-3-carboxylic acid (33). Methyl iodide(350 TL, 1.16 mmol) and K₂CO₃ (320 mg, 2.32 mmol) were added to asolution of 32 (152 mg, 0.58 mmol) in DMF (4 mL) and stirred at rt underAr for 14 h. Upon completion, ethyl acetate was added and the reactionwas washed with 1% HCl (2×20 mL), brine (1×), dried over MgSO₄ andconcentrated in vacuo and carried onto the next step without furtherpurification.

LiOH.H₂O (60 mg, 1.43 mmol) was added to a solution of fully methylateddiflunisal (140 mg, 0.50 mmol) in MeOH:THF:H₂O (4.5 mL 1:1:1), andstirred at rt for 4 h. Upon completion, the reaction was acidified with30% HCl, extracted with ethyl acetate (3×5 mL), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(2:1 ethyl acetate:hexane, 1% acetic acid) to afford 33 (122 mg, 93%) asa white solid. ¹H NMR (CDCl₃, 400 MHz) δ 10.77 (br s, 1H), 8.31 (dd, 1H,J=2.5, 0.9 Hz), 7.75 (dt, 1H, J=8.6, 2.1 Hz), 7.41 (dt, 1H, J=8.9, 6.6Hz), 7.15 (d, 1H, J=8.8 z), 6.94 H (m, 1H), 4.13 (s, 3H). ¹³C NMR(CDCl₃, 100 MHz) δ 177.7, 161.4, 158.2, 135.7, 133.9, 131.4, 129.0,123.5, 118.1, 112.2, 112.0, 104.6, 56.9. HRESIMS calculated forC₁₄H₁₀F₂O₃ (M−H) 263.0520, found 263.0514. Normal phase HPLC retentiontime: 21.6 min. Reverse phase HPLC retention time: 11.9 min. >99% pure.

Compounds 34–38 were prepared according to Scheme 7. Compounds 39–44were prepared according to Scheme 8. To a solution of aryl iodide (1.0equiv) in enough toluene to give a concentration of 0.07 M, was added anappropriate formyl phenylboronic acid dissolved in enough EtOH toprovide a concentration of 0.4 M boronic acid. A 2 M aqueous solution ofNa₂CO₃ was added to give a final reaction concentration of 0.04 M withrespect to aryl iodide, followed by addition of Pd(PPh₃)₄ (3.0 mol %).The reaction was heated to reflux under Ar for 18 h, and uponcompletion, was cooled to rt and extracted with CH₂Cl₂ (2×), washed withbrine (1×), dried over MgSO₄ and concentrated in vacuo. The residue waspurified by flash chromatography (40:1 hexane:ethyl acetate) to affordthe biphenyl aldehydes as white solids in 40–91% yields.

3′,5′-Dichloro-3-formylbiphenyl (39). ¹H NMR (CDCl₃, 400 MHz) δ 10.09(s, 1H), 8.04 (t, 1H, J=1.8 Hz), 7.91 (dt, 1H, J=7.6, 1.3 Hz), 7.80(ddd, 1H, J=7.8, 2.0, 1.3 Hz), 7.64 (t, 1H, J=7.8 Hz), 7.49 (d, 2H,J=1.8 Hz), 7.38 (t, 1H, J=1.9 Hz). ¹³C NMR (CDCL₃, 100 MHz) δ 192.0,142.8, 139.7, 137.2, 135.8, 133.0, 130.1, 130.0, 128.1, 128.0, 125.9.HRFABMS calculated for C₁₃H₈Cl₂O (M+H) 251.0027, found 251.0027. Normalphase HPLC retention time: 8.0 min. Reverse phase HPLC retention time:15.2 min. >99% pure.

3′,5′-Dichloro-4-formylbiphenyl (40). ¹H NMR (CDCl₃, 400 MHz) δ 7.99(AA′XX′, 2H, J_(AA′)=J_(XX′)=2.1 Hz, J_(XA)=J_(X′A′)=8.5 Hz,J_(X′A)=J_(XA′)=0.7 Hz, ν_(A)=ν_(A′)=3193.7 Hz, ν_(X)=ν_(X′)=3077.8 Hz),7.70 (AA′XX′, 2H, as above), 7.47 (t, 1H, J=1.9 Hz), 7.39 (d, 2H, J=1.9Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 191.8, 144.4, 142.9, 136.2, 135.8,130.6, 128.5, 127.9, 126.1. HREIMS calculated for C₁₃H₈Cl₂O (M−H)248.9873, found 248.9874. Normal phase HPLC retention time: 7.9 min.Reverse phase HPLC retention time: 15.2 min. >99% pure.

3′,5′-Dichloro-2-formylbiphenyl (41). ¹H NMR (CDCl₃, 400 MHz) δ 9.98 (s,1H), 8.03 (dd, 1H, J=7.8, 1.3 Hz), 7.66 (td, 1H, J=7.6, 1.5 Hz), 7.55(tt, 1H, J=7.6, 1.0 Hz), 7.44 (t, 1H, J=1.9 Hz), 7.39 (dd, 1H, J=7.7,1.0 Hz), 7.27 (d, 2H, J=1.9 Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 191.4,142.9, 141.0, 135.3, 134.0, 133.7, 130.7, 129.0, 128.5, 128.4, 128.4.HRFABMS calculated for C₁₃H₈Cl₂O (M+H) 251.0030, found 251.0029. Normalphase HPLC retention time: 7.0 min. Reverse phase HPLC retention time:14.9 min. >99% pure.

Compounds 45–47 were prepared according to Scheme 8. To a solution ofbiphenyl aldehyde (1.0 equiv) in enough acetone to give a concentrationof 0.07 M, was added KMnO₄ (2.0 equiv) in enough H₂O to give aconcentration of 0.2 M permanganate. The reaction was stirred for 16 hat rt, and upon completion, was concentrated in vacuo and the resultingresidue was redissolved in 10:1 CH₂Cl₂:MeOH and filtered through a plugof glass wool. The crude product was purified by flash chromatography(10:1 CH₂Cl₂:MeOH) to afford the carboxylic acids (58 mg, 100%) as whitesolids in 82–100% yields.

2′,4′-Dichlorobiphenyl-3-carboxylic acid (45). ¹H NMR (DMSO-d₆, 400 MHz)δ 8.22 (br s, 1H), 8.00 (br s, 1H), 7.94 (d, 1H, J=7.5 Hz), 7.76 (s,2H), 7.63 (s, 1H), 7.60 (br s, 1H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 168.0,143.7, 138.1, 135.4, 131.6, 129.9, 127.9, 126.2. HRESIMS calculated forC₁₃H₈Cl₂O₂ (M−H) 264.9823, found 264.9810. Normal phase HPLC retentiontime: 12.3 min. Reverse phase HPLC retention time: 14.2 min. >99% pure.

2′,4′-Dichlorobiphenyl-4-carboxylic acid (46). ¹H NMR (CD₃OD, 400 MHz) δ8.11 (br s, 2H) 7.72 (m, 2H), 7.64 (d, 2H, J=1.9 Hz), 7.46 (t, 1H, J=1.7Hz). ¹³C NMR (DMSO-d₆, 100 MHz) δ 170.3, 140.6, 135.2, 127.2, 126.4,119.3, 118.6, 117.4. HRESIMS calculated for C₁₃H₈Cl₂O₂ (M−H) 264.9830,found 264.9823. Normal phase HPLC retention time: 12.5 min. Reversephase HPLC retention time: 14.4 min. >99% pure.

2′,4′-Dichlorobiphenyl-2-carboxylic acid (47). ¹H NMR (DMSO-d₆, 400 MHz)δ 7.75 (br s, 1H), 7.56 (s, 2H), 7.48 (m, 2H), 7.36 (m, 2H). ¹³C NMR(DMSO-d₆, 100 MHz) δ 170.1, 152.5, 145.2, 133.3, 130.0, 129.6, 128.0,127.2, 126.3. HRESIMS calculated for C₁₃H₈Cl₂O₂ (M−H) 264.9823, found264.9834. Normal phase HPLC retention time: 11.4 min. Reverse phase HPLCretention time: 13.6 min. >99% pure.

Compounds 48–53 were prepared according to Scheme 8. To a solution ofbiphenyl aldehyde (1.0 equiv) in enough MeOH to give a concentration of0.1 M, was added NaBH₄ (2.0 equiv) in enough MeOH to give aconcentration of 0.3 M borohydride. The reaction was stirred at 0° C.,and slowly warmed to rt, and after stirring for 16 h, was concentratedin vacuo and purified by flash chromatography (3:1 hexane:ethyl acetate)to afford the biphenyl alcohols as a white solids in 94–100% yields.

3′,5′-Dichlorobiphenyl-3-yl-methanol (48). ¹H NMR (CDCl₃, 400 MHz) δ7.54 (m, 1H), 7.46 (d, 2H, J=1.8 Hz), 7.45 (m, 2H), 7.39 (m, 1H), 7.34(t, 1H, J=1.9 HZ), 4.77 (s, 2H), 1.90 (br s, 1H). ¹³C NMR (CDCl₃, 100MHz) δ 144.1, 141.9, 139.0, 135.5, 129.5, 127.4, 127.1, 126.5, 125.8,125.7, 65.3. HREIMS calculated for C₁₃H₁₀Cl₂O (M+) 252.0103, found252.0109. Normal phase HPLC retention time: 13.9 min. Reverse phase HPLCretention time: 14.0 min. >99% pure.

3′,5′-Dichlorobiphenyl-4-yl-methanol (49). ¹H NMR (CDCl₃, 400 MHz) δ7.53 (AA′XX′, 2H, J_(AA′)=1.9 Hz, J_(XX′)=3.1 Hz, J_(XA)=8.7 Hz,J_(X′A′)=6.4 Hz, J_(X′A)=J_(XA′)=0.5 Hz, ν_(A)=ν_(A′)=3009.8 Hz,ν_(X)=ν_(X′)=2977.8 Hz), 7.45 (AA′XX′, 2H, as above), 7.45 (d, 2H, J=1.9Hz), 7.33 (t, 1H, J=1.9 Hz), 4.75 (br d, 2H, J=4.8 Hz), 1.81 (br t, 1H,J=5.2 Hz). ¹³C NMR (CDCl₃, 100 MHz) δ 144.0, 141.4, 138.0, 135.5, 127.8,127.4, 127.4, 125.8, 65.1. HREIMS calculated for C₁₃H₁₀Cl₂O (M+)251.0110, found 252.0109. Normal phase HPLC retention time: 15.4 min.Reverse phase HPLC retention time: 14.0 min. >97% pure.

3′,5′-Dichlorobiphenyl-2-yl-methanol (50). ¹H NMR (CDCl₃, 400 MHz) δ7.55 (dd, 1H, J=7.5, 1.3 Hz), 7.43 (td, 2H, J=7.5, 1.4 Hz), 7.38 (m,2H), 7.29 (d, 2H, J=1.9 Hz), 7.24 (dd, 1H, J=7.4, 1.4 Hz), 4.58 (s, 2H),1.79 (s, 1H). ¹³C NMR (CDCl₃, 100 MHz) δ 143.7, 138.9, 137.9, 134.9,130.0, 129.0, 128.9, 128.2, 127.9, 127.6, 63.0. HREIMS calculated forC₁₃H₁₀Cl₂O (M+) 252.0110, found 252.0109. Normal phase HPLC retentiontime: 11.5 min. Reverse phase HPLC retention time: 14.0 min. >99% pure.

Compounds 54 and 55 were prepared according to Scheme 9. To a solutionof the appropriate iodobenzaldehyde (1.0 equiv) in enough toluene togive a concentration of 0.07 M, was added 3,5-difluorophenyl boronicacid (2.0 equiv) dissolved in enough EtOH to provide a concentration of1.0 M boronic acid. A 2 M aqueous solution of Na₂CO₃ was added to give afinal reaction concentration of 0.04 M with respect to iodobenzaldehyde,followed by addition of Pd(PPh₃)₄ (4.0 mol %). The reaction was stirredat 60° C. for 17 h, and upon completion, was cooled to rt and extractedwith CH₂Cl₂ (2×), washed with brine (1×), dried over MgSO₄ andconcentrated in vacuo. The residue was purified by flash chromatography(10:1 hexane:ethyl acetate) to afford the biphenyl aldehydes as whitesolids in 78–80% yields.

3′,5′-Difluoro-3-formylbiphenyl (54). ¹H NMR (CDCl₃, 400 MHz) δ 10.06(s, 1H), 8.02 (t, 1H, J=1.4 Hz), 7.88 (dt, 1H, J=7.8, 1.4 Hz), 7.78(ddd, 2H, J=7.8, 2.0, 1.2 Hz), 7.61 (t, 2H, J=7.7), 7.10 (m, 2H), 6.80(tt, 1H, J=8.8, 2.3 Hz. ¹³C NMR (CDCl₃, 100 MHz) δ 192.0, 164.8, 162.3,143.0, 139.8, 137.1, 132.9, 129.9, 127.9, 110.4, 103.5. HRFABMScalculated for C₁₃H₈F₂O (M+H) 219.0620, found 219.0621. Normal phaseHPLC retention time: 8.9 min. Reverse phase HPLC retention time: 13.7min. >99% pure.

3′,5′-Difluoro-4-formylbiphenyl (55). ¹H NMR (CDCl₃, 400 MHz) δ 9.98 (s,1H), 8.02 (dd, 1H, J=7.8, 1.5 Hz), 7.65 (td, 1H, J=7.3, 1.4 Hz), 7.54(t, 1H, J=7.8 Hz), 7.40 (dd, 1H, J=7.6, 1.2 Hz), 6.90 (m, 3H). ¹³C NMR(CDCl₃, 100 MHz) δ 191.5, 164.1, 161.6, 143.4, 141.3, 134.0, 133.7,130.6, 129.0, 128.3, 113.3, 103.8. HRFABMS calculated for C₁₃H₈F₂O (M+H)219.0620, found 219.0621. Normal phase HPLC retention time: 7.0 min.Reverse phase HPLC retention time: 13.4 min. >99% pure.

A number of in vitro tests can be used to evaluate the compounds fortheir ability to stabilize transthyretin tetramers or prevent formationof fibrils. The tests can include a fibril formation assay, a plasmaselectivity assay, determination of the three-dimensional structure of atransthyretin:compound complex (e.g. by X-ray crystallography), kineticsof transthyretin tetramer dissociation or fibril formations, anddetermining the stoichiometry and energetics of transthyretin:compoundinteractions, by, for example, centrifugation or calorimetry. Details ofexemplary in vitro assays are presented below.

Each compound was subjected to a stagnant fibril formation assay.Compounds were dried over P₂O₅ overnight and dissolved in DMSO to afinal concentration of 7.2 mM to provide a primary stock solution (10×stock). A secondary stock solution was prepared by five-fold dilution ofthe primary stock solution with DMSO to a final concentration of 1.44 mM(2× stock). The acid-mediated amyloidogenicity of TTR (3.6 TM) in thepresence of inhibitors (1.44 mM) was measured as follows: To adisposable UV cuvette were added 495 TL of a 0.4 mg/mL WT TTR proteinsolution in 10 mM sodium phosphate, 100 mM KCl and 1 mM EDTA (pH 7.6)and 5 TL of the 1.44 mM secondary stock inhibitor solution in DMSO (2×stock). The mixture was vortexed and incubated for 30 min (25° C.), atwhich time the pH was lowered to 4.4 with 500 TL of 200 mM acetate, 100mM KCl and 1 mM EDTA (pH 4.2). The final 1 mL solution was vortexed andincubated for 72 h at 37° C. without agitation. After 72 h, the cuvetteswere vortexed to suspend any fibrils present, and the turbidity of thesuspension was measured at 350 and 400 nm using a UV-vis spectrometer.The percent fibril formation was obtained by the ratio of the observedturbidities for each TTR plus inhibitor sample relative to that of asample prepared the same way, but lacking inhibitor, multiplied by 100.The fibril formation assay employing equimolar inhibitor and TTRconcentrations (3.6 TM) was performed as above using a 1× secondarystock solution. The 1× stock solution was prepared by ten-fold dilutionof the 7.2 mM 10× primary stock solution with DMSO to a finalconcentration of 0.72 mM and used in the fibril formation assay asdescribed above. All assays were performed in triplicate and allcompounds were assayed using wild-type TTR. All compounds were found tobe soluble throughout the course of the experiment by testing theturbidities of the solutions in the absence of WT TTR, ensuring thatturbidity was the result of TTR amyloid formation.

The binding stoichiometries of potential inhibitors to TTR in bloodplasma were evaluated by an antibody capture/HPLC method. A 1.5-mLeppendorf tube was filled with 1.0 mL of human blood plasma and 7.5 TLof a 1.44 mM DMSO solution of the inhibitor under evaluation. Thesolution was incubated and gently rocked at 37° C. for 24 h. A 1:1gel:TSA (Tris saline) slurry (125 TL) of quenched sepharose was added tothe solution and gently rocked at 4° C. for 1 h. The solution wascentrifuged (16,000×g) and the supernatant was divided into two 400 TLaliquots, which were then added to different 200 TL samples of a 1:1gel:TSA slurry of the anti-TTR antibody-conjugated sepharose. Thesolutions were gently rocked at 4° C. for 20 min, centrifuged(16,000×g), and the supernatant was removed. The gel was washed with 1mL of TSA/0.05% saponin (3×, 10 min each) at 4° C., followed by 1 mL ofTSA (2×, 10 min each) at 4° C. The samples were centrifuged (16,000×g),the final wash was removed, and 155 TL of 100 mM triethylamine, pH 11.5,was added to elute the TTR and bound inhibitors from the antibodies.After gentle rocking at 4° C. for 30 min, the elution sample wascentrifuged (16,000×g) and 145 TL of the supernatant, containing TTR andinhibitor, were removed. The supernatant was then analyzed byreverse-phase HPLC as described previously. See, for example, Purkey, H.E.; Dorrell, M. I.; Kelly, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,5566–71, which is incorporated by reference in its entirety.

Crystals of WT TTR were obtained from protein solutions at 7 mg/mL (in100 mM KCl, 1 mM EDTA, 10 mM sodium phosphate, pH 7.0, 0.35–0.50 Mammonium sulfate) equilibrated against 2 M ammonium sulfate in hangingdrops. The TTR-ligand complexes were prepared from crystals soaked formore than three weeks with a 10-fold molar excess of the ligand. ACCD-PXL-L600 detector (Bruker instruments) coupled to an RU200 rotatinganode X-ray generator was used for data collection of crystals soakedwith 20 or 26. The Quantum-4 detector at the monochromatic high-energysource of 14-BM-C, BIOCARS, Advance Photon Source was used for the datacollection of crystals soaked with 1 or 18. The crystals were placed inparatone oil as a cryo-protectant and cooled for diffraction experiments(120 K for 20 and 26, and 100 K for 1 and 18). Crystals of TTR•ligandcomplex structures are isomorphous with the apo crystal form with unitcell dimensions close to a=43 Å, b=85 Å, and c=66 Å; space group P2₁2₁2with two monomers in the asymmetric unit. Data sets of 1 and 18 werereduced with DENZO and SCALEPACK. See Otwinowski, Z.; Minor, W.Macromolecular Crystallography, Part A, in Methods in Enzymology;Carter, C. W., Sweet, R. M., Eds.; Academic Press: 1997; Vol. 276, p307–326, which is incorporated by reference in its entirety. Data setsof 20 and 26 were reduced with SAINT and PROSCALE (Bruker AXS, Inc.).

The protein atomic coordinates for TTR from the Protein Data Bank(accession number 1BMZ) were used as a starting model during themolecular replacement search by EPMR. The best solutions from EPMR wererefined by molecular dynamics and energy minimization protocols of CNS.The resulting difference Fourier maps revealed binding of the ligands(in two conformations for 18, 20 and 26, and four conformations for 1)in each binding pocket of the TTR tetramer. Using these maps, the ligandcould be unambiguously placed into the density and was included in thecrystallographic refinement. After several cycles of simulated annealingand subsequent positional and temperature factor refinement, watermolecules were placed into difference Fourier maps. The final cycle ofmap-fitting was done using the unbiased weighted electron density mapcalculated by the shake/warp bias removal protocol. All bindingconformations of the ligand were in good agreement with unbiasedannealed omit maps as well as the shake/warp unbiased weighted mapsphased in the absence of the inhibitor. Final cycles of the refinementwere carried out by the restrained refinement protocol of Refmac.Because of the lack of interpretable electron densities in the finalmap, the nine N-terminal and three C-terminal residues were not includedin the final model. A summary of the crystallographic analysis ispresented in Table 2. See, for example, Kissinger, C. R.; Gehlhaar, D.K.; Fogel, D. B. Acta Crystallogr., Sect. D 1999, 55, 484–491; Brunger,A. T.; et al. Acta Crystallogr., Sect. D 1998, 54, 905–921;Kantardjieff, K.; et al. Acta Crystallogr., Sect. D 2002, 58, 735–743;Bailey, S. Acta Crystallogr., Sect. D 1994, 50, 760–763; and Murshudov,G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., Sect. D 1997, 53,240–255, each of which is incorporated by reference in its entirety.

The kinetics of TTR tetramer dissociation was evaluated by linkedmonomer unfolding in urea. Slow tetramer dissociation is not detectableby far-UV CD spectroscopy, but is linked to the rapid (500,000-foldfaster) unfolding step easily detectable by far-UV CD as describedpreviously. TTR tetramer (3.6 TM) dissociation kinetics as a function ofinhibitor (3.6 TM) were evaluated by adding 3.6 TL of a 1 mM solution(in ethanol) of the inhibitor of interest to 69 TL of WT TTR (2.90mg/mL, 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH 7.0) to whichwas added 127.4 TL of phosphate buffer. For an inhibitor concentration(7.2 TM) twice that of the TTR concentration (3.6 TM), 7.2 TL of a 1 mMsolution (in ethanol) of the inhibitor of interest was added to 69 TL ofWT TTR (2.90 mg/mL, 10 mM sodium phosphate, 100 mM KCl, 1 mM EDTA, pH7.0) to which was added 123.8 TL of phosphate buffer. 100 TL of theprotein-inhibitor solution of interest was added to a solution of 600 TLof 10.3 M urea and 300 TL of phosphate buffer, to yield a final ureaconcentration of 6.5 M. The solutions were vortexed and the circulardichroism spectra were collected at the following intervals: 0, 5, 8,23, 46, 71, 95, 118, 144 and 168 h. A control sample containing 7.2 TLof ethanol rather than inhibitor was prepared for comparison and thespectra were collected at the time points identified above. CD spectrawere collected between 220 and 213 nm, with scanning every 0.5 nm and anaveraging time of 10 sec. Each wavelength was scanned once. The valuesfor the amplitude were averaged between 220 and 213 nm to determine theextent of θ-sheet loss throughout the experiment.

The rate of acid-mediated fibril formation was followed at pH 4.4 byturbidity. Compounds were dried over P₂O₅ overnight and dissolved inDMSO to a final concentration of 7.2 mM to provide a primary stocksolution (10× stock). A secondary stock solution was prepared byfive-fold DMSO dilution of the primary stock solution to yield a finalconcentration of 1.44 mM (2× stock). The fibril formation assayemploying an inhibitor concentration of 7.2 TM relative to 3.6 μM TTR(tetramer) was performed as follows: To a disposable UV cuvette wereadded 495 TL of a 0.4 mg/mL WT TTR protein solution in 10 mM sodiumphosphate, 100 mM KCl and 1 mM EDTA (pH 7.6) and 5 TL of the 1.44 mMsecondary inhibitor stock solution (2× stock). The mixture was vortexedand incubated for 30 min (25° C.). After 30 min, the pH was lowered to4.4 with 500 TL of 200 mM acetate, 100 mM KCl, 1 mM EDTA (pH 4.2). Thefinal 1 mL solution was vortexed and incubated at 37° C. withoutagitation. The solutions were vortexed and turbidity at 350 and 400 nmwas measured. UV spectra were collected at the following intervals: 0,4, 8, 24, 48, 72, 96, 120, 144, 168 and 192 h after acidification. Acontrol sample containing 5 TL of DMSO was prepared for comparison, andthe spectra were collected at the time points above. Each inhibitorsolution was prepared in groups of 10 to prevent disturbance of thecuvettes before a reading was taken. After a UV absorbance was obtained,the cuvettes corresponding to that time-point were discarded. The fibrilformation assay employing equimolar (3.6 TM) TTR and inhibitorconcentration was performed as above using a 1× secondary inhibitorstock solution prepared as follows: A stock solution was prepared byten-fold dilution of the 7.2 mM 10× primary stock solution with DMSO toa final concentration of 0.72 mM and used in the fibril formation assayas described above. All compounds were found to be soluble throughoutthe course of the experiment, ensuring that turbidity was the result ofTTR amyloid formation.

The TTR quaternary structure in the presence of inhibitors at pH 4.4 wasanalyzed. The mechanism by which 18 and 20 stabilize TTR was evaluatedby incubating the protein (3.6 μM) for 72 h under the conditions of thestagnant fibril formation assay in the presence of either 3.6 μM or 7.2μM inhibitor. After 72 h, the samples were centrifuged (14,000×g) andthe supernatant was removed from any solid that was formed in the assay.Equilibrium and velocity ultracentrifugation analysis was achieved witha Beckman XL-I analytical ultracentrifuge. The acquisition and analysisof data was performed as described previously. See, for example,Lashuel, H. A.; Lai, Z.; Kelly, J. W. Biochemistry 1998, 37, 17851–64;and Lashuel, H. A.; et al. Biochemistry 1999, 38, 13560–73, each ofwhich is incorporated by reference in its entirety.

The dissociation constants characterizing the binding of 18 and 20 to WTTTR were determined by isothermal titration calorimetry using a Microcalinstrument (Microcal Inc., Northhampton, Md.). A solution of inhibitor(300 μM or 500 μM in 25 mM tris buffer, 100 mM KCl, 1 mM EDTA, 12% EtOH,pH 8.0) was prepared and titrated into an ITC cell containing WT TTR (15μM or 25 μM in 25 mM tris buffer, 100 mM KCl, 1 mM EDTA, 12% EtOH, pH8.0). The initial injection of 2.5 μL was followed by 50 injections of5.0 μL each (25° C.). Integration of the thermogram after subtraction ofblanks yielded a binding isotherm that fit best to a model of twosequential binding sites with negative cooperativity. The data were fitby a nonlinear least squares approach with four adjustable parameters,namely, K₁, ΔH₁, K₂, ΔH₂ using the ITC data analysis module in ORIGINversion 2.9 provided by Microcal.

The compounds described were evaluated as TTR amyloid fibril inhibitorsusing a turbidity assay. WT TTR amyloidosis was initiated byacidification of TTR preincubated with inhibitor (25° C., 30 min),employing buffer addition to jump the pH to a final value of 4.4. Afterincubation of each mixture for 72 h (37° C.), the turbidity was measuredat 350 and 400 nm using a UV-vis spectrometer. All amyloid fibrilformation data was normalized to WT TTR amyloidogenesis in the absenceof inhibitor, assigned to be 100% fibril formation. Therefore, 5% fibrilformation corresponds to a compound inhibiting 95% of WT TTR fibrilformation after 72 h. Each potential inhibitor was first evaluated at aconcentration of 7.2 μM relative to a TTR tetramer concentration of 3.6TM. Compounds allowing less than 15% fibril formation were reevaluatedat a concentration equal to the TTR concentration (3.6 TM) to select forthe inhibitors with the highest efficacy. Fibril formation of less than40% under these conditions is characteristic of a very good inhibitor,whereas 40–70% inhibition is indicative of a modest compound. Fibrilformation data is presented in Table 1.

TABLE 1 Effects of Compounds on Fibril Formation Plasma Compound %fibril formation % fibril formation Selectivity Number Structure (3.6 μMinhibitor) (7.2 μM inhibitor) (equiv bound) diflunisal(1)

37.0 3.4 0.13 ± 0.02  2

31.5  3

32.4  4

46.3  5

53.1  6

19.5  7

19.6  8

40.9 10.2 0.18 ± 0.05  9

16.4 10

61.2 11

39.4 9.1 none observed 12

32.6 2.6 0.20 ± 0.05 13

15.7 14

13.3 15

39.4 9.8 none observed 16

32.4 4.8 0.08 ± 0.00 17

35.7 5.6 0.23 ± 0.00 18

35.7 3.7 1.27 ± 0.12 19

35.1 6.7 0.29 ± 0.12 20

28.5 4.5 0.50 ± 0.05 21

30.8 22

51.5 14.3 0.08 ± 0.01 23

38.7 2.6 0.09 ± 0.00 24

38.7 2.5 0.07 ± 0.02 25

35.5 1.0 none observed 26

29.9 3.6 0.27 ± 0.02 27

47.4 15.4 none observed 28

38.5 3.5 none observed 29

31.7 3.4 0.07 ± 0.02 30

25.5 4.4 0.12 ± 0.02 31

25.8 3.8 0.26 ± 0.04 32

69.9 33

38.5 34

100.0 35

100.0 36

99.4 37

100.0 38

52.2 39

30.6 4.4 1.30 ± 0.15 40

25.4 41

34.5 7.1 1.96 ± 0.11 42

35.4 43

93.5 44

72.5 45

32.7 3.0 0.80 ± 0.08 46

41.2 4.9 1.56 ± 0.01 47

45.4 48

30.0 3.3 0.89 ± 0.09 49

38.9 5.9 0.54 ± 0.10 50

33.6 7.7 none observed 51

85.5 52

100.0 53

81.0 54

64.3 55

69.6

Based on the inhibitor efficacy data for compounds 2–55, it appears thata carboxylate-substituted hydrophilic ring directly connected to adi-halogen functionalized hydrophobic ring is sufficient for excellentactivity (Table 1). A phenolic substituent in lieu of a carboxylate(2–10) yields considerably less active inhibitors, far inferior to theparent compound 1. Inhibitors having a halogen in the ortho or metaposition of the hydrophobic ring are superior to compounds lackinghalogens or those having a single para halogen. This suggests that parahalogens do not compliment the HBPs in the same manner as meta and orthohalogenated biaryls. Complete removal of all halogens can result in apoor inhibitor, presumably due to the lack of steric complimentarity tofill the halogen binding pockets (for example, compounds 10, 21–22, 27,42–44, and 51–53). Under the conditions tested, the best phenoliccompound (8) is inferior to 1, which bears both a phenolic andcarboxylate functionality on the hydrophilic ring. Biaryl compoundsstabilized with a single carboxylate (such as 11–22) can be excellentamyloid fibril inhibitors, for example, compounds 11, 12, 15–20. Theserival diflunisal for inhibition, the exception being those containingonly a para halogen (e.g., compounds 13 and 14). A meta or parasubstituted aryl carboxylate can be sufficient for endowing excellentinhibition properties, suggesting that the hydroxyl substituent in 1 isnot required for good inhibitor activity. In addition, para carboxylatepositioning appears to afford superior inhibitors, suggesting that apara carboxylate is better able to take advantage of electrostaticinteractions with the ε-ammonium groups of Lys 15 and 15′ (forwardbinding mode), as in the case of 20, or hydrogen bonding interactionswith the Ser 117 and 117′ hydroxyl groups (reverse binding mode) as inthe case of 18. Biaryls wherein the hydrophobic ring is substituted withhalogens in positions other than the para position and the hydrophilicring with meta and particularly para carboxylates yield highlyefficacious TTR amyloid fibril formation inhibitors.

Addition of a hydroxyl substituent to the ring containing a carboxylatesubstituent (the salicylic acid substitution, for example, 23–27) canalso result in inhibitors with high activity similar to diflunisal. Inbiaryls with the salicylic acid core, the exact positioning of thehalogens does not appear to be as vital as in the previous cases,suggesting that this ring contributes disproportionately to the bindingenergy. The para hydroxyl may participate in hydrogen bonding with theε-ammonium groups of Lys 15 and 15′ (forward binding mode) or with theSer 117 and 17′ hydroxyls (reverse binding mode). Substitution offluorine in 1 with chlorine (26) can result in an inhibitor with equalor superior activity, whereas complete removal of the halogens (27) canresult in a modest inhibitor. It should be noted that 27 is onlyslightly superior to a para carboxylate 22 in vitro, and both aresuperior to the halogen-free inhibitors with the carboxylate in the metaposition, 21, and the hydroxyl-containing analog 10.

Inclusion of a 3′,5′-dihalo-4′-hydroxyl substituent on thehalogen-containing ring, with carboxylates in either the para or metapositions (28–31) can result in high inhibitory activity, similar todiflunisal. The 4-hydroxyl substitution was included to more closelymimic thyroxine, the natural ligand of TTR. These inhibitors may alsomore closely mimic the hormone activity of thyroxine and therefore mayact as thyroid agonists or antagonists, an effect that can beundesirable.

Protection of the carboxylate as a methyl ester or the hydroxyl as amethyl ether (32 and 33) can result in inferior inhibitors comparedto 1. A combination of the loss of charge and the increase in stericbulk probably explains these observations. Removal of all hydrophilicsubstituents (e.g., 34–38) can result in poor inhibitors. A biarylcompound containing only meta chlorine substitution (e.g., 38) can be amodest inhibitor, suggesting that the chlorines make enhanced contactsin the halogen binding pockets as compared to fluorine-containingbiaryls (37).

Several chlorine-containing inhibitors were synthesized and their TTRfibril inhibition activity evaluated. When members of this class ofinhibitors contain carboxylates in the meta or para positions (e.g., 45and 46) they can possess high activity, whereas those having an orthocarboxylate (such as 47) can be an inferior inhibitor. This observationsuggests that the ortho carboxylate may be too far from the Lys 15 and15′ ε-ammonium groups to make favorable electrostatic interactions(forward binding mode) or from the Ser 117 and 117′ hydroxyl groups toundergo hydrogen bonding interactions (reverse binding mode). Benzylicalcohols 48–50 surprisingly proved to be excellent inhibitors of fibrilformation. The meta dichloro substitution on one ring appears to becomplemented by benzyl alcohol functionality in either the ortho, metaor para position, potentially due to the hydrogen bonding orwater-mediated hydrogen bonding. A series of aldehyde analogs (39–41)where the —CH₂OH groups were replaced by an aldehyde functionality,showed good inhibition except in the case of the para aldehyde 41,possibly owing to hydration of the aldehyde to a gem diol. It ispossible that the aldehydes, the benzylic alcohols and the carboxylatesbind in the pocket via a different mechanism. In the absence ofstructural information, however, a similar binding mode cannot be ruledout. It is also possible that the aldehydes bind covalently either toSer 117 (117′) via a hemiacetal or to Lys 15 (15′) via an imine bond.Substitution of the chlorines with fluorines (54 and 55) in the case ofthe aldehydes can result in rather poor inhibitors (39 and 41). Asbefore, complete removal of the halogens can result in inhibitors withpoor activity (42 and 44), except in the case of the meta aldehyde 43where the activity is modest. This modest activity may result from ahigh degree of hydration. It is surprising that the 3′,5′-difluoro-metaaldehyde (54), is inferior to the aldehyde lacking halogens (42).

Inhibitors that keep TTR fibril formation below 50% at a concentrationequal to that of TTR (3.6 μM) were further evaluated for their abilityto bind TTR selectively over all other proteins in blood plasma. Thediflunisal concentration in blood can exceed 30 μM 20 h after a single500 mg dose, or 300 μM 4 h after the same dose. While this high level ofsustained plasma concentration suggests excellent bioavailability, moreselective inhibitors will allow for lower dosing and potentially fewerside-effects; therefore, human plasma was incubated with this subset ofinhibitors at a final concentration of 10.8 μM (average TTRconcentration in human plasma is approximately 5 μM). TTR was thencaptured using a resin-bound antibody, and the immobilized TTR waswashed three times with a solution of TSA (tris saline)/0.05% saponin,followed by two washes with TSA. The TTR-inhibitor complex was liberatedfrom the resin with 100 mM triethylamine (pH 11.5), and thestoichiometry of inhibitor present relative to TTR was determined byreverse-phase HPLC analysis. A maximum of 2 equiv of inhibitor may bebound per TTR tetramer. The post-wash plasma binding stoichiometries,representing lower limits owing to wash-associated losses, aresummarized in Table 1.

Chlorine-containing biphenyls can be selective for binding TTR in humanblood plasma (average stoichiometry of 0.8, with a theoretical maximumstoichiometry of 2.0, see Table 1). The average stoichiometry observedwas 0.4 for all inhibitors tested. Of the fluorine-containinginhibitors, 18 and 20 exhibited very good and acceptable bindingselectivity for TTR, respectively, superior to the 0.13 stoichiometrydisplayed by 1 under similar conditions. The stoichiometry valuesreported in Table 1 can represent a lower limit due to wash-associatedlosses of the inhibitor from the TTR sequestered on a polyclonalantibody resin. The TTR binding selectivity results for 39 and 41 shouldbe considered with caution, because these compounds may be covalentlyattached to TTR, as discussed above.

Those inhibitors that exhibit excellent TTR amyloid fibril inhibitiondata in vitro, yet display poor plasma selectivity, may bindpreferentially to the drug-binding sites in albumin and/or similar sitesin other proteins found in plasma. It can be unlikely that suchinhibitors will prevent TTR misfolding and amyloidosis in a complexenvironment like that of blood plasma or CSF.

High-resolution X-ray co-crystal structures of 1 and three of itsanalogs 26, 18, and 20 bound to TTR were obtained by soaking TTRcrystals with a 10-fold molar excess of inhibitor for more than threeweeks. The crystallographic statistics are summarized in Table 2.

TABLE 2 Crystallographic Statistics Data collection TTR•1 TTR•18 TTR•20TTR•26 Resolution (Å) 35.58–1.85 42.18–1.54 64.5–1.7 51.30–1.7 No ofunique 20,478 33,741 25,634 25,486 reflections Completeness (%)98.4/99.0 95.0/98.0 98.0/99.0 99.0/98.0 (Overall/outer shell) R_(sym)(Overall/ 0.09/0.31 0.03/0.32 0.08/0.39 0.07/0.40 outer shell)Refinement Resolution (Å) 35.58–1.85 42.18–1.50 64.5–1.7 51.30–1.7R-factor/R-free (%) 21.2/23.6 22.2/24.5 22.5/24.0 21.5/24.2 Rmsd bond0.03 0.06 0.02 0.02 length (Å) Rmsd bond 2.3 2.7 1.9 1.9 angles (°)

Diflunisal (1) binds to TTR in both forward and reverse modes. In eachhormone-binding site of TTR, four different binding conformations ofdiflunisal were found with approximately equal occupancy—a forward andreverse binding mode each with two symmetrically equivalent bindingmodes. The biaryl system of diflunisal was shifted away from the centerof the hormone binding pocket and occupies two distinct positions toform a ‘V’ shaped cone of electron density in the hormone-binding pocketof TTR. This mode of binding enhances both hydrophobic and van der Waalsinteractions between the inhibitor and the hydrophobic pocket of TTRformed by Leu17, Ala 108, Leu 110, Thr 119 and Val 121. The reversebinding mode of diflunisal was augmented by the hydrogen bondinteraction between the carboxyl group and the side chain oxygen of Thr119 and the main chain oxygen of Ala 108 in the inner binding pocket.Surprisingly Ser 117 neither takes up multiple conformations nor formsany electrostatic interaction with the inhibitor. In the reverse mode ofbinding, one of the fluorine substituents of diflunisal was withinhydrogen bonding distance from the Thr 119 side chain oxygen (3.3 Å). Inthe outer binding pocket, the electron density for the side chain atomsof Lys 15′ was visible only at low sigma level indicating it may be inmore than one conformation. The best possible conformation for the Lys15 residue was modeled at a hydrogen bonding distance from the carboxylgroup of diflunisal in the forward binding mode.

Compound 20 binds to TTR in the forward binding mode, with thecarboxylate-substituted hydrophilic ring oriented in the outer bindingpocket to interact electrostatically with Lys 15 and 15′. Thefluorinated aryl ring is positioned in the inner binding pocket whereinthe halogens are placed in HBP 2 and 2′. Interestingly, close inspectionof both binding sites reveals a significant difference in theorientation of the biphenyl rings. The angles between the planes of thephenyl rings vary from 32.6 degrees in one binding site to 63.8 degreesin the other. This observation may be a result of the negativelycooperative binding of 20 with TTR.

Compound 18 binds to TTR in the reverse mode with thecarboxylate-substituted hydrophilic aryl ring oriented into the innerpocket, within hydrogen bonding distance of Ser 117 and Ser 117′. Thearyl rings are rotated 34 degrees with respect to one another to takeadvantage of hydrophobic interactions with Leu 17, Ala 108, Val 121 andThr 119. The fluorines are positioned in halogen binding pockets 1 and1′. The reverse binding mode was not expected, instead, the carboxylatewas envisioned to be positioned in the outer pocket to take advantage ofelectrostatic interactions with Lys 15 and 15′, with the fluorinessequestered into halogen binding pockets 2 and 2′. However, the reversebinding mode was not a total surprise, as it was observed previously fordiclofenac (a biaryl amine) and several diclofenac analogs.

Substitution of chlorines in place of fluorines in diflunisal inducessignificant differences in the binding of 26 to TTR. Compound 26 bindsto TTR in the reverse binding mode with the carboxyl-substituted arylring oriented in the inner binding pocket and chlorines sequestered intohalogen binding pockets 2 and 2′. Like 18 and 20, compound 26 alsooccupies the center of the hormone-binding pocket. The residues Ala 108,Lys 15, Leu 17, Leu 110, Lys 17 and Thr 119 of TTR protomers forms vander Waals and hydrophobic interactions with the inhibitor. In the innerbinding pocket, the side chain of Ser 117 exists in two conformations tointeract with the carboxyl substitution of 26 and Ser 117 of the othermonomers. The same carboxyl oxygen of 26 also forms a hydrogen bondinteraction with the main chain oxygen of Ser 117. The other carboxyloxygen of 26 forms a hydrogen bond with the main chain oxygen of Ala108. In contrast to diflunisal, the Thr 119 residue orients away fromthe inhibitor, contributing to the hydrophobicity of the binding pocketrather than hydrogen bonding with the inhibitor.

To further probe the mechanism of action of these inhibitors, theirability to stabilize TTR against urea-induced dissociation as a functionof time was evaluated. The rate of tetramer dissociation was linkedirreversibly to fast, easily monitored, monomer unfolding employing ureaconcentrations exceeding those that enable monomer refolding.Unfolding-monitored dissociation was probed by far UV-CD in 6.5 M urearevealing that all the good inhibitors of acid-mediated amyloidogenesisslowed the rate of tetramer dissociation in a dose-dependent fashion(FIGS. 2A and 2B). Several inhibitors, including 20, 46 and 48, show adramatic effect on dissociation of the TTR tetramer, the rate-limitingstep of amyloidogenesis. See, for example, Hammarstrom, P.; et al. Proc.Natl. Acad. Sci. U.S.A. 2002, 25, 16427–32, which is incorporated byreference in its entirety.

Since the mode of inhibition of TTR fibril formation by these compoundsis suspected to be dose-dependent tuning of the tetramer dissociationbarrier through ground-state stabilization, the best inhibitors shouldslow tetramer dissociation the most. The rate of fibril formation wasmonitored by turbidity at a final pH of 4.4 over 192 h. See FIGS. 3A,3B, 4A and 4B. Inhibitors possessing the ability to stabilize tetramericTTR at low pH will prevent tetramer dissociation, misfolding andmisassembly into amyloid. The best inhibitors of amyloid fibrilformation are those that slow tetramer dissociation the most (FIGS. 2Aand 2B). however, the correlation is not perfect, as some inhibitorsbind better in urea than in acidic conditions and vice versa.

To ensure that the inhibitors are stabilizing the tetrameric form of TTR(3.6 μM), the quaternary structure of TTR was probed with equilibriumand velocity analytical ultracentrifugation studies. The quaternarystructure of the protein after 72 h incubation with 18 and 20 (3.6 μM or7.2 μM) at pH 4.4 was determined. The tetramer was the dominant species,both at 3.6 μM and 7.2 μM inhibitor concentration in equilibrium AUC aswell as in velocity studies.

Isothermal titration calorimetry (ITC) was employed to determine thebinding constants of 18 and 20 to TTR at pH 8.0 (25° C.). Diflunisal andthe two analogs bind to TTR with negative cooperativity, acharacteristic displayed by many other ligands. Binding at the firstsite is 15 times stronger than binding at the second site in the case ofdiflunisal and 20. Biaryl 18 possesses a K_(d1) approximately 120 timeslower than K_(d2) (Table 3). Table 3 summarizes the first and seconddissociation constants for the binding of 1, 18 and 20 to wild type TTRdetermined by ITC. The binding constants for 1 were reported previously,and are provided here for comparison purposes. See Example 1.

TABLE 3 Dissociation Constants for Compounds Binding to Wild Type TTRInhibitor K_(d1) K_(d2) 1 75 nM 1100 nM 18  9 nM 1100 nM 20 80 nM 1300nM

Tetrameric WT TTR dissociates with a t_(1/2) of 42 h, and unfolds500,000 times faster. Hence, its rate of dissociation can be probed bylinking it to unfolding, which is irreversible in 6.5 M urea. Sincetetramer dissociation is rate-limiting for amyloidogenesis, allinhibitors displaying excellent in vitro activity and bindingstoichiometry exceeding 0.50 in plasma should slow tetramer dissociationif the presumed mechanism of action, kinetic stabilization by selectivebinding to the native state, is correct (see FIGS. 2A and 2B).

TTR tetramer dissociation rates were measured as a function of inhibitorconcentration over a 168 h time-course in 6.5 M urea. Select inhibitors,specifically 18, 20, 39, 41, 45, 46, 48 and 49 demonstrate an overallreduction in the extent of tetramer dissociation over 160 h as reflectedin the amplitude changes relative to TTR without inhibitor. The rate oftetramer dissociation is also dramatically slowed in the presence ofinhibitor, as reflected in the decrease in the slope of the time course.Inhibitors 20, 45, 46 and 48 are superior, presumably because theinhibitor dissociates very slowly from TTR·I and TTR·I₂ due to theirhigh binding affinity in urea. The formation of TTR·I and TTR·I₂ cansignificantly stabilize the native state due to the low K_(d)s of suchcomplexes, and raise the kinetic barrier for tetramer dissociation,substantially in the case of 20, 45, 46 and 48. Even though 16 and 18bind to TTR, it appears that their affinity is insufficient to affectkinetic stabilization. It is likely significant that the rank orderingof inhibitor efficacy in urea at an inhibitor concentration of 3.6 μM(3.6 μM protein) is 20≈45>46≈48, which is different than an inhibitorconcentration of 7.2 μM (20≈46>45≈48). This likely reflects a differencein the K_(d2) values in urea.

Kinetic stabilization of the native state is an attractive therapeuticstrategy due to the emerging evidence that misfolded oligomers, whetheron the amyloid pathway or off it, are neurotoxic. Achieving kineticstabilization with inhibitors can provide a non-invasive treatment forSSA, FAP and FAC.

Tetramer dissociation rates in urea in the presence of a given inhibitordo not always predict the ability of the inhibitor to preventamyloidosis at low pH. Since it is not yet clear how and where amyloidforms in a human, TTR tetramer stabilizers that function well in avariety of denaturing environments are desirable. The rate of TTR fibrilformation as a function of inhibitor concentration was explored underacidic conditions (FIGS. 3A, 3B, 4A and 4B). Inhibitors 20, 45 and 48perform exceptionally well in this environment as well. Inhibitor 46 isa better tetramer stabilizer in urea than in acid, whereas 1 is muchbetter in acid than in urea. The free energy of stabilization associatedwith the formation of the TTR·I and TTR·I₂ complexes in a givenenvironment determines the extent of ground-state stabilization andassociated increase in activation free energy for tetramer dissociation.These data suggest that the inhibitors slow TTR amyloidosis at low pHmuch more efficiently than they slow TTR tetramer dissociation in 6.5 Murea. This may be because amyloidogenesis requiresconcentration-dependent reassembly after dissociation. The moreeffective inhibitors are those that can keep the concentration of themonomeric amyloidogenic intermediate of TTR at low enough levels to makefibril formation very inefficient. As observed in the urea denaturationof TTR in the presence of inhibitors, the rank ordering of inhibitorefficacy at low pH differs significantly from 3.6 μM inhibitor (FIGS. 3Aand 3B) to 7.2 μM inhibitor concentration (FIGS. 4A and 4B). Thisobservation likely reflects the differences in K_(d2) values of each ofthe inhibitors at low pH. The most dramatic example is that ofdiflunisal—one of the most efficacious inhibitors of fibril formation at3.6 μM, but one of the least efficacious at 7.2 μM, owing to itsrelatively high K_(d2).

The diflunisal analogs represent a promising class of compounds for thetreatment of TTR amyloidosis. While several diclofenac analogs are verygood inhibitors of fibril formation, the diflunisal analogs offer anadditional class of highly effective TTR tetramer stabilizers. Severaldiclofenac analogs offer the ability to inhibit fibril formationresulting from the dissociation and misfolding of two TTRmutants—Val30Met and Leu55Pro. X-ray co-crystal structures demonstratethat the diclofenac analogs primarily bind in the reverse binding mode,however, minor perturbations in the structures of the diflunisal analogsallow for either forward or reverse binding. In addition, diflunisal isable to bind either in the forward or the reverse binding mode, withalmost equal occupancy in both modes. The dissociation constantsobtained for diclofenac (60 nM for K_(d1) and 1200 nM for K_(d2)) werecomparable to those obtained for diflunisal and 20, with 18demonstrating nearly 10-fold tighter binding for the first binding eventas illustrated by its K_(d1) value. In addition, both inhibitor classesdisplayed negatively cooperative binding. Most remarkably, severaldiflunisal analogs were very selective for TTR in human blood plasma,offering the potential for decreased toxicity and side-effects. See Oza,V. B.; et al. J. Med. Chem. 2002, 45, 321–32.

Twenty eight of the compounds synthesized can substantially inhibit TTRamyloidogenesis. Of those, several showed binding stoichiometryexceeding 0.50 equiv in human blood plasma. Both the chlorinated andfluorinated aryl substructures of the better inhibitors are found inknown drugs, therefore, there is good reason to believe that thesecompounds or their analogs could be evolved into drugs that do notdisplay the NSAID activity of 1. The fluorinated compounds 18 and 20 canbind to and stabilize tetrameric TTR in 6.5 M urea, dramatically slowingthe first step of misfolding and amyloidogenesis, dissociation of theTTR tetramer. These compounds, and others, also dramatically slowacid-mediated TTR amyloidogenesis. Of the compounds tested, 18, 20, 39,41, 45, 46, 48 and 49 performed best at stabilizing the TTR tetramer inurea and under acidic conditions. These biaryl compounds appear toincrease the activation barrier associated with tetramer dissociation,the rate-limiting step for amyloid formation, by ground-statestabilization.

Example 3 Orally Administered Diflunisal Stabilizes TransthyretinAgainst Denaturation

Transthyretin (TTR) is a homotetrameric protein that transportsthyroxine and holo-retinol binding protein. Under denaturing conditions,rate limiting tetramer dissociation and rapid monomer misfolding enablesmisassembly into amyloid-causing senile systemic amyloidosis, familialamyloid polyneuropathy, and familial amyloid cardiomyopathy. Diflunisalbinding to at least one of the two unoccupied thyroxine binding sites inTTR is known to stabilize the TTR tetramer also increasing thedissociation activation barrier in vitro. The feasibility of usingdiflunisal for the treatment of TTR amyloidosis was investigated.

Methods

Thirty healthy volunteers (25 male, 5 female) were enrolled afterinformed consent was given. The subjects ranged from 23 to 53 years ofage (mean age, 37.6±8.8) with a mean body weight of 78.0±12.1 kg. Eachsubject was treated with diflunisal (Dolobid®) at a dose of 125, 250 or500 mg twice a day (every 12 hrs) for 7 days (13 total doses). Blood wascollected on day 1 before treatment and on day 8, 4 and 12 h afterdiflunisal intake. This study design was approved by the Human SubjectsCommittee of Scripps Clinic, Scripps Green Hospital, The ScrippsResearch Institute, and The Scripps General Clinical Research Center.

Serum diflunisal levels were measured. One hundred μL of serum was addedto 900 μL of acetonitrile to precipitate the proteins. Followingcentrifugation, 100 μL of supernatant was added to 900 μL of 100 mMaqueous triethylamine, pH 11.5. After filtration, 100 μL of each samplewas injected on a Keystone 3-cm C18 reverse-phase column using a 40–100%gradient of solution B over 10 min (solution A: 94.8% water/5%acetonitrile/0.2% trifluoroacetic acid; solution B: 94.8%acetonitrile/5% water/0.2% trifluoroacetic acid), controlled by a Waters600E multisolvent delivery system. Detection was accomplished at 280 nmwith a Waters 486 tunable absorbance detector, and the peaks wereintegrated to give the concentration of diflunisal from standard curves.

Stoichiometry of diflunisal binding to TTR in human serum was analyzed.A 1:1 gel/10 mM Tris.HCl, pH 8.0/140 mM NaCl/0.025% NaN₃(TSA) slurry(62.5 μL) of Sepharose was added to 500 μL of serum and incubated at 4°C. for 1 h. Following centrifugation, 400 μL of supernatant was added to200 μL of a 1:1 gel/TSA slurry of the anti-TTR antibody-conjugatedSepharose and slowly rocked at 4° C. for 20 min. After centrifugation,the gel was washed with 1 mL of TSA/0.05% saponin (Fisher Scientific)(twice, 10 min each), and additionally with 1 mL of TSA (once, 10 min)at 4° C. Then 155 μL of 100 mM aqueous triethylamine, pH 11.5, was addedto elute the TTR and bound diflunisal from the antibodies. After gentlerocking at 4° C. for 30 min, the sample was centrifuged and 145 μL ofthe supernatant was removed. A 135-μL injection of sample was separatedand analyzed as described previously (Purkey et al., Proc Natl Acad SciUSA 2001; 98: 5566–71).

Serum TTR tetramer stability towards urea denaturation was evaluated.Ten μL samples of serum were incubated (25° C.) in 90 μL of variousconcentrations of urea in 50 mM phosphate buffer (pH 7.0; 100 mM KCl, 1mM EDTA, 1 mM DTT). Urea solutions were checked by refractive index toverify the concentrations prepared by weight. Glutaraldehydecross-linking of the protein was performed by adding 10 μL ofglutaraldehyde (25%). The cross-linking reaction was allowed to proceedfor 4 min before it was quenched by the addition of 10 μL of NaBH₄ (7%in 0.1 M NaOH). The samples were mixed with 120 μL of SDS reducing gelloading cocktail (final SDS concentration=2.5%) and boiled for 5 min.Samples were separated using 12% SDS-PAGE and the gels were analyzed byimmunoblotting using anti-TTR antiserum (Purkey et al., supra).

Serum TTR tetramer stability against acid denaturation was evaluated.Ten μL samples of serum were incubated (37° C.) in 90 μL of 100 mMacidification buffer. Citrate buffer was used when a final pH of ≦3.8was desired; acetate buffer was employed when the pH range underevaluation was 4.2–5.4. After cross-linking, samples were analyzed bySDS-PAGE and immunoblotting as described above.

Recombinant WT TTR and variants were expressed in BL21/DE3 Epicuriangold Escherichia coli (Stratagene) transformed with the pmmHα plasmidcontaining the TTR and ampicillin-resistance genes. Expression andpurification were performed as described previously (Lashuel et al.,Biochemistry 1999; 38: 13560–73).

Rate of TTR tetramer dissociation was measured by circular dichroismspectroscopy. The evaluation of tetramer dissociation rates was carriedout using recombinant TTR (3.6 μM) samples in 6.5 M urea, aconcentration in the post-transition region for tertiary structuralchange (Hammarstrom et al., Proc Natl Acad Sci USA 2002; 99: 16427–32).The far-UV CD spectra of TTR (210–220 nm) as a function of time wasmeasured to evaluate the slow tetramer dissociation rate by linking itto fast tertiary structural changes.

Fibril formation assay was carried out as follows. A recombinant TTRstock solution (7.2 μM) was diluted 1:1 with 100 mM acidificationbuffer. Citrate buffer was used when a final pH of ≦3.8 was desired;acetate buffer was employed when the pH range under evaluation was4.2–6.0, and phosphate buffer was utilized for evaluatingamyloidogenesis at pH 6.5. Samples were incubated at 37° C. for 72 hwithout stirring after acidification. The extent of fibril formation wasprobed by turbidity measurements at 400 mm.

Fibril formation kinetics were measured as follows. Solutions ofrecombinant TTR (7.2 μM) were mixed with an equal volume of 100 mMacetate buffer to yield a final pH of 4.4. The samples were incubated at37° C. and the turbidity at 400 nm was monitored over the course of 168h. A separate sample was made up for each time point.

The effect of diflunisal on urea-mediated tetramer dissociation andpH-mediated fibril formation was evaluated by adding diflunisal to a TTRsolution which was incubated for 3 h (37° C.) before subjecting theprotein to urea denaturation or pH-mediated amyloidosis.

Results

The mean serum diflunisal concentrations, measured by HPLC, 4 and 12 hafter intake of the 13^(th) dose were 20.1±7.1 and 6.9±3.0 μM in the 125mg bid group, 233.5±76.0 and 145.8±38.9 μM in the 250 mg bid group, and517.0±79.5 and 421.9±78.1 μM in the 500 mg bid group. Greater than 99%of diflunisal is protein bound. These concentrations observed in 250 mgbid and 500 mg bid group are very high relative to the TTR concentrationin serum (3.6–7.2 μM) and should yield a diflunisal bindingstoichiometry approaching the maximum of 2 if binding to competitorproteins such as TBG (0.3–0.5 μM) and/or albumin (580–725 μM), which hasmultiple binding sites for small molecules, is not of high affinity.

Diflunisal preferably binds tetrameric TTR in blood with stoichiometryof at least 1 and ideally 2 to observe maximum kinetic stabilization. Toplace a lower limit on diflunisal stoichiometry in each subject, weimmunoprecipitated transthyretin from serum with polyclonal antibodiesbound to a solid phase resin as described previously (Purkey et al.,supra). After washing immobilized TTR 3× to eliminate non-specificbinding, the TTR-diflunisal complex was dissociated from the resin andthe diflunisal stoichiometry was determined by HPLC employing standardcurves. The stoichiometry of diflunisal bound to TTR in serum 4 and 12 hafter intake was 0.45±0.11 and 0.31±0.12 in the 125 mg bid group,1.12±0.08 and 0.95±0.13 in the 250 mg bid group and 1.51±0.09 and1.48±0.08 in the 500 mg bid group. Diflunisal stoichiometry increasedwith its serum concentration, up to ≈300 μM. The lower than expectedmaximal stoichiometry of 1.5 at a serum concentration of 300 μM eitherresults from a limitation of the method (wash-associated losses) and/ordiflunisal binding to other plasma proteins, therefore we carried out adiflunisal binding stoichiometry study with recombinant TTR.Wash-associated losses explain the maximum binding stoichiometry of 1.5owing primarily to dissociation from the low affinity site. Diflunisalbinds to TTR with negative cooperativity, hence dissociation from thelow affinity site is dramatically faster. The expected bindingstoichiometry in buffer was calculated based on the dissociationconstants determined by isothermal titration calorimetry (K_(d1), 75 nM;K_(d2), 1.1 μM). Coplotting the calculated and experimentally determinedstoichiometry, the latter derived from immunoprecipitation (3 washes)and HPLC analysis, allows one to estimate the true stoichiometry at1.75–1.91 at 250 mg bid, suggesting that this dose could be utilized.

A comparison of diflunisal (100 μM) binding stoichiometry in subjects(0.8–1) to recombinant TTR (1.5) reveals significant binding to serumproteins besides TTR, providing the incentive to develop diflunisalanalogs that bind more selectively to TTR. The serum level of TTR wasincreased and the serum levels of total T₄ and RBP were decreased afterdiflunisal administration in all groups. These findings suggest thatdiflunisal influences TTR metabolism. No obvious side effects wereobserved during or after the study. However, the serum level of albuminwas decreased significantly and the levels of BUN and creatinine wereincreased slightly in the 500 mg bid group. In the 250 mg bid group, theserum level of albumin was decreased moderately and the level of BUN wasincreased slightly.

A new method was developed to demonstrate that orally administereddiflunisal stabilizes serum TTR against denaturation stresses includingamyloidosis. This method serves as a surrogate marker to identifycompounds that should prevent TTR misfolding diseases. Whole serum fromthe subjects was subjected to denaturation either by adding urea (0–9 M)or by adding acid (pH 3.8–5.4). Since TTR must dissociate in order todenature, quaternary structural changes can be used to monitor theextent of unfolding (Hammarstrom et al, supra). Glutaraldehyde was addedto crosslink all the proteins in serum after being subjected to adenaturation stress and to establish what fraction of TTR is normallyfolded (tetramer or dimer) versus denatured (monomer). SDS-PAGE of wholeserum separates the crosslinked TTR tetramer and dimer (theserepresenting folded TTR) from the monomer. Immunoblotting enablesquantitative comparisons of the amount of folded TTR. The polyclonalantibodies do not bind to the unfolded TTR monomer nearly as well asfolded TTR, therefore it is most useful to compare the intensity of thetetramer and dimer bands in the absence and presence of diflunisal. Thetime dependence of diflunisal inhibition of TTR denaturation can also beevaluated by this method. The barely noticeable time dependence of thisprocess in the presence of diflunisal strongly supports the kineticstabilization mechanism (see Example 1) wherein ground statestabilization by diflunisal makes the tetramer dissociation barrierinsurmountable. The efficacy of the diflunisal (250 mg bid) in thesedenaturation time courses is better than the measured stoichiometry(0.8–1.2) would predict, providing further evidence that theimmunoprecipitation method underestimates the actual bindingstoichiometry, especially when it exceeds 1.

Knowing the range of diflunisal binding stoichiometries in humans andthe concentration of diflunisal required to mimic those stoichiometriesin a test tube allows for the carrying out of relevant in vitrobiophysical studies to probe the mechanism by which the TTR•diflunisaland TTR•diflunisal₂ complexes prevent dissociation and amyloidosis. Therate of urea-mediated (6.5 M) dissociation and the rate of acid mediated(pH 4.4) amyloid fibril formation were studied as a function ofdiflunisal concentration (5, 10, 20 and 60 μM), revealing dose dependentslowing. Since tetramer dissociation is rate limiting for amyloid fibrilformation, it follows that tetramer dissociation rates in urea should bepredictive of the extent of amyloid fibril formation mediated byacidification. Diflunisal is better at inhibiting amyloidosis thaninhibiting urea mediated dissociation because concentration dependentreassembly is also required for amyloidosis. It is also possible thatK_(d1) and K_(d2) associated with diflunisal binding to TTR are lower inacid than in urea.

More than 80 TTR mutations predispose individuals to hereditaryamyloidosis by sequence dependent alterations of the denaturation energylandscape. Of these, the amyloid deposition of V122I results in familialamyloid cardiomyopathy (FAC) in 3–4% of African Americans, whereas V30Mis the prominent familial amyloid polyneuropathy (FAP) mutation.Diflunisal inhibits both V122I and V30M amyloidogenesis in a dosedependent fashion, demonstrating the generality of this approach.

It is highly desirable to develop a general, non-invasive therapeuticstrategy to ameliorate TTR amyloidosis. The results outlined hereinindicate that oral administration of diflunisal can slow tetramerdissociation by binding to and stabilizing the non-amyloidogenic nativestate. Native state stabilization is a particularly attractive strategygiven recent reports that misfolded oligomers and not amyloid fibrilscause neurodegeneration. Clinical use of diflunisal (250–500 mg bid) forrheumatoid arthritis and osteoarthritis demonstrate its low toxicity forlong-term uses. TTR's serum half-life is 12–15 h, therefore twice dailydosing seems optimal given the 8–10 h half-life of diflunisal.Diflunisal should be effective against SSA, FAC and FAP, because itbinds both WT and variant TTR imposing kinetic stabilization, analogousto mechanism utilized by the inclusion of trans-suppressor subunits intoTTR tetramers otherwise composed of disease-associated subunits, whichis known to ameliorate human disease. Diflunisal may be less effectiveagainst CNS amyloidosis because it cannot cross the blood-brain barrier,although diflunisal analogs (e.g., an analog described herein) may havesuch an ability.

Example 4 Hydroxylated Polychlorinated Biphenyls Selectively BindTransthyretin in Blood and Inhibit Amyloidogenesis

Polychlorinated biphenyls (PCBs) are known persistent environmentalpollutants that are reported to be toxic to rodents and possibly humans.The longevity of these compounds in the environment is due to their slowdegradation and high lipophilicity, which allows them to bioaccumulateand concentrate as they move up the food chain. Hydroxylated PCBs(OH-PCBs) are metabolites formed by oxidation of PCBs by the P450monooxygenases. Definitive data on the toxicity of individual PCBcompounds in humans is difficult to find due to the fact that thecommercially available PCBs are generally mixtures that contain manydifferent isomers as well as trace amounts of known toxins, e.g.chlorinated dibenzofurans. However, the toxicity of several purifiedPCBs has been demonstrated in laboratory animals. Bone loss, immunologictoxicity, neurotoxicity and lowered thyroid hormone levels, in additionto the estrogenicity of the OH-PCBs are associated with theadministration of these compounds.

Numerous studies demonstrate that PCBs and OH-PCBs bind to transthyretin(TTR) in vitro. It has been suggested that TTR is the protein target inhuman blood that contributes to the persistence of the OH-PCBs inexposed individuals. While numerous reports suggest TTR as a PCB bindingprotein in vivo, there is no definitive evidence that PCBs bind totransthyretin in plasma. We have developed an immunoprecipitation methodthat can be used to place a lower limit on the binding stoichiometry ofsmall molecules to TTR in biological fluids. The TTR bindingstoichiometry of PCBs and OH-PCBs to human plasma TTR was evaluatedherein.

Post-secretion amyloidogenesis of plasma TTR requiring rate limitingtetramer dissociation, monomer misfolding and misassembly putativelycauses senile systemic amyloidosis, familial amyloid cardiomyopathy andthe familial amyloid polyneuropathies. Herein, several OH-PCBs aredemonstrated to bind selectively to TTR in human plasma and inhibitamyloid fibril formation through tetramer stabilization leading topartial or complete kinetic stabilization of the native state. Fourrepresentative TTR•(OH-PCB)₂ complexes were characterized by x-raycrystallography to better understand the molecular basis for binding andto provide the basis for the design of optimized TTR amyloidogenesisinhibitors.

Binding Selectivity of PCBs and OH-PCBs for Transthyretin in Human BloodPlasma

The binding selectivity of eight PCBs (compounds 1–8, FIG. 5), reportedto displace thyroid hormone from TTR with an IC₅₀ of less than 50 nM andfourteen OH-PCBs (compounds 9–22, FIG. 6), known PCB metabolites thatare reported to bind to TTR or lower thyroxine levels in mice or ratswere evaluated. Lower limits on PCB binding stoichiometry to TTR inplasma were established using polyclonal TTR antibodies covalentlyattached to a sepharose resin that was mixed with human blood plasmapretreated with PCB or OH-PCB (10.8 μM). After washing, the PCB orOH-PCB binding stoichiometry to TTR (≈5 μM) was evaluated by reversephase HPLC.

Up to two PCBs can bind to the two identical thyroid hormone bindingsites in a TTR tetramer. Except for PCBs 1 & 3, the remainingnon-hydroxylated PCBs displayed relatively low binding selectivity forplasma TTR (Table 4). In contrast, the OH-PCBs showed good to excellentbinding selectivity for plasma TTR (Table 5). Several of thehydroxylated PCBs (e.g., 16 and 22) approach a binding stoichiometry of2. The binding selectivity of OH-PCBs in whole blood is very similar tothat observed in plasma, hence erythrocyte membranes do notsignificantly sequester the OH-PCBs studied.

TABLE 4 Binding Stoichiometry of PCBs to TTR in Human Blood PlasmaCompound Equivalents Bound 3 1.50 ± 0.42 1 0.62 ± 0.12 6 0.19 ± 0.11 20.18 ± 0.03 5 0.06 ± 0.04 4 0.05 ± 0.04 7 No Binding 8 No Binding

TABLE 5 Binding Stoichiometry of Hydroxylated PCBs to TTR in Human BloodPlasma Compound Equivalents Bound (Plasma) Equivalents Bound (Blood) 161.86 ± 0.14 ND 22 1.67 ± 0.40 1.69 17 1.63 ± 0.05 ND 19 1.48 ± 0.16 1.5521 1.40 ± 0.22 1.33 18 1.36 ± 0.21 ND 12 1.23 ± 0.24 1.47 11 1.12 ± 0.221.20 20 1.02 ± 0.09 0.86 10 0.96 ± 0.09 0.93 13 0.84 ± 0.24 0.86 9 0.83± 0.19 0.57 14 0.81 ± 0.29 0.73 15 0.70 ± 0.17 0.56

The antibody capture of the TTR•PCB complex has the potential tounderestimate the PCB binding stoichiometry owing to PCB dissociationfrom TTR during the 5 wash steps. PCBs and OH-PCBs (10.8 μM) wereincubated with recombinant TTR (3.6 μM) to evaluate the stoichiometry ofsmall molecule bound to immobilized TTR after each wash step.Stoichiometry decreased by 10–17% for PCB 2 and OH-PCB 18 after 5washes, whereas that of PCB 4 decreased by 45%. Quantification ofwash-associated losses allows one to estimate the true bindingstoichiometry of PCBs and OH-PCBs in plasma. Furthermore, a goodcorrelation between the final stoichiometry of OH-PCB bound torecombinant TTR and the amount bound to TTR in plasma indicates that thecompound is a highly selective TTR binder in plasma, e.g., OH-PCB 18. Incontrast, PCBs 2 and 4 exhibit a higher binding stoichiometry to TTR inbuffer than in plasma, strongly suggesting that they bind to competitorprotein(s) as well as TTR in plasma.

TTR Amyloid Fibril Inhibition by Hydroxylated PCBs

The ability of OH-PCBs and PCB 3 to inhibit TTR fibril formation invitro was evaluated because these compounds exhibit good bindingselectivity to TTR in blood. TTR secreted into blood from the liverappears to be the source of systemic TTR amyloid. While it is not yetclear where or how amyloid is formed in humans, the typical denaturantin cells is acid, which is effective in converting nearly allamyloidogenic peptides and proteins into amyloid and/or relatedaggregates. Hence, acid-mediated (pH 4.4) fibril formation monitored byturbidity was employed to monitor the effectiveness of the PCBs asinhibitors. Hydroxylated PCBs and PCB 3 were highly efficacious as TTRfibril inhibitors. At an inhibitor concentration equal to the WT TTRconcentration (3.6 μM), only 12–50% of the normal amount of fibrilformation was observed after a 72 h incubation period. This activity isequivalent to that displayed by the best fibril inhibitors discovered todate, such as flufenamic acid (Flu), which was included as a positivecontrol.

Binding of OH-PCB 18 to TTR

Previous mass spectrometry experiments suggest that OH-PCB 18 exhibitspositive binding cooperativity to TTR's two C₂ related thyroid hormonebinding sites. When substoichiometric (<1:1) amounts of 18 are added toTTR, the predominant species observed in the mass spectrometer areapo-TTR and the TTR•18₂ complex, consistent with positively cooperativebinding. The TTR binding characteristics of 18 are in contrast to thoseexhibited by numerous other TTR amyloid fibril inhibitors that bind withnegative cooperativity. Isothermal titration calorimetry studies carriedout under physiological conditions reveal that the binding of OH-PCB 18to WT TTR fits best to a model where the dissociation constants areidentical K_(d)s (3.2±1.8 nM). This result does not disprove positivelycooperative binding, as one cannot achieve a low enough concentration ofTTR to probe positive cooperativity because of the insufficient heatreleased. Attempts to fit the collected data to models of positively ornegatively cooperative binding yielded poor fits.

Co-Crystal Structures of OH-PCBs 12, 16, 17 and 18

Crystals of OH-PCBs 12, 16, 17 and 18 bound to WT TTR were obtained bysoaking TTR crystals with a 10-fold excess of inhibitor for four weeks.X-ray structures were then solved for each of the complexes. The TTRdimer within the crystallographic asymmetric unit forms half of the twoligand-binding pockets. Because both binding sites are bisected by thesame two-fold axis of symmetry, two symmetry equivalent binding modes ofthe inhibitors are typically observed. Each TTR binding site can besubdivided into inner and outer cavities. These cavities comprise threeso-called halogen binding pockets (HBPs) because they are occupied bythe iodines on the two aromatic rings of thyroxine. HBP 3 and 3′ arelocated deep within the inner binding cavity, HBP 2 and 2′ define theboundary between the inner and outer binding cavity, whereas HBP 1 and1′ are located near the periphery of outer binding cavity. Theco-crystal structures reveal that the C—C bond connecting the twoaromatic rings of the OH-PCB are nearly centered on the 2-fold symmetryaxis, giving the appearance of a single binding conformation. Thedihedral angle between two phenyl rings is 59° for 12, 37° for both 16and 17, and 44° for 18. All of the OH-PCBs occupy similar positions inthe inner and outer binding pockets. The van der waals complimentarilyof the biaryl ring system facilitates several inter-subunit interactionsinvolving residues X, Y and Z in one subunit and residues n′, m′, and o′in the other subunit composing each binding site. Several of thesubstituents on the phenyl rings are off-axis and can be modeled inmultiple positions within the observed electron density.

OH-PCB 18 Bound to TTR

The 1.8 Å X-ray structure of the TTR•18₂ complex demonstrates that theinhibitor has excellent steric complementarity with the TTR bindingsite. Molecular mechanics (Insight II, Accelrys) indicates that theunbound conformation of 18 is close to its bound structure. The refinedstructure defines direct and water-mediated electrostatic interactionsthat contribute to high affinity binding of 18. One of the 3-Cl, 4-OH,5-Cl identically substituted aromatic rings occupies the inner bindingpocket, its chlorine substituents projecting into HBP 3 and 3′. The sidechains of Ser117 and Thr119 adopt an alternative conformation byrotation about their Cα-Cβ bonds as discerned by the unbiased electrondensity maps. The side chain of Ser117 adopts all three rotomerconformations as discerned by the distribution of electron density.Interestingly, two water molecules are located in between the adjacentSer117 residues at the two-fold axis with 50% occupancy, facilitating anetwork of hydrogen bonds connecting the Ser117 residues, the nearbywater molecules and the phenol functionality of 18. It is not clear froman inspection of the structure why 18 binds with non- or positivelycooperative behavior. The other identically substituted ring occupiesthe outer TTR binding pocket with its halogens projecting into HBPs 1and 1′.

Compound 16 Bound to TTR

The 3-Cl, 4-OH, 5-Cl tri-substituted phenolic ring of 16 is orientedinto the inner binding site of TTR making the same electrostatic andhydrophobic interactions with TTR that this ring does in the TTR•18₂structure described above. The 3,4-dichlorinated aromatic ring occupiesthe outer binding pocket, with the halogen directed into HBP-1 or 1′depending upon which symmetry equivalent binding mode is beingconsidered. The electron density of 16, like that of OH-PCB 18, issymmetric and thus it is not possible to position the para OH and paraCl unambiguously based upon the electron density map. The unbiasedelectron density map is consistent with three rotomer conformations ofSer117 and contains two water molecules in between the Ser117 residues,analogous to the TTR•18₂ structure.

OH-PCB 17 Bound to TTR

Inhibitor 17 binds with its 3-Cl, 4-OH, 5-Cl substituted aryl ringoriented into the inner binding pocket utilizing the same interactionsthat this ring uses in the TTR•16₂ and TTR•18₂ structures describedabove. The 2,3,4-tri-chlorinated ring occupies the outer binding pocketutilizing interactions with HBP-1, HBP-1′, HBP-2, HBP-2′in the twosymmetry equivalent binding modes. The multiple conformations of Ser117and the two conserved water molecules are also features of the TTR•17₂structure. A conformational change of the Thr119 side chain was evidentfrom the unbiased electron density maps.

Compound 12 Bound to TTR

Biaryl 12 places its 3-Cl, 4-OH substituted aryl ring in the outerbinding pocket, with its two chlorines interacting with HBP-1 and 1′. Incontrast to the structures of TTR•16₂ and TTR•17₂ where the phenol islocated in the inner binding pocket. The hydroxyl group (probably in theionized form) is within hydrogen bonding distance of the Lys15 sidechains. The tetra-chlorinated ring is placed in the inner binding pocketwherein the halogens are oriented in HBPs 2 and 2′ as well as 3 and 3′.The Ser117 and Thr119 side chains adopt conformations that are identicalto those found in the apo-TTR structure, unlike the situation in 16, 17and 18.

Herein, of 8 PCBs previously reported to displace T4 with an IC₅₀ ofless than 50 nM, only 1 and 3 were shown to bind to TTR with anappreciable stoichiometry in human plasma. In contrast, all fourteenOH-PCBs previously reported to bind to TTR exhibited significant bindingselectivity to TTR in plasma. This is consistent with the observationthat OH-PCBs are observed primarily in plasma and appear to beselectively retained there, as opposed to retention in lipids and othertissues where PCBs typically accumulate. The OH-PCBs also bindselectively to TTR in whole blood consistent with the idea that they donot partition into lipid membranes.

The amount of PCB (or OH-PCB) that washes off of the antibody•TTR•PCBcomplex during the washing steps was evaluated using recombinant WT TTR.The extent of wash-associated PCB dissociation is molecule specific.Some compounds exhibit high binding stoichiometry after the washes,consistent with significant initial binding and low wash-associatedlosses, implying a slow dissociation rate. Compounds exhibiting lowbinding stoichiometry fall into at least two categories: high initialbinding stoichiometry with significant wash-associated losses or lowinitial binding stoichiometry without significant wash-associatedlosses, the latter scenario applicable to compounds that bind with highaffinity to TTR, but with even higher affinity to another plasmaprotein(s). PCBs 2 and 4 both exhibit low post-wash bindingstoichiometry to recombinant TTR. Forty five % of PCB 4 was lost due towashes whereas PCB 2 simply exhibits poor initial binding stoichiometrywith minimal wash-associated losses (10%). The post-wash selectivityvalues reflect a lower limit of the amount of PCB that is initiallybound in plasma. Compounds like PCB 18, which are characterized by highpost-wash binding stoichiometry must have high binding affinity andselectivity, consistent with the slow off rate observed.

In addition to their high binding selectivity to plasma TTR, the OH-PCBsand PCB 3 also display excellent inhibition of TTR fibril formation invitro. The efficacy of inhibitors 14, 15, and 18 are among the highestobserved to date at equimolar inhibitor and TTR concentration (3.6 μM).This is likely attributable to their high binding affinity (alsoconsistent with their low off rate) and their non- or positivelycooperative TTR binding properties which are unusual. The nM K_(d)sexhibited by the best inhibitor, OH-PCB 18, dictates that the nativestate of TTR will be stabilized by >3 kcal/mol. Ground statestabilization raises the tetramer dissociation barrier (rate limitingstep in TTR amyloidogenesis) substantially, such that the tetramercannot dissociate on a biologically relevant timescale. Kineticstabilization of the native non-amyloidogenic state mediated by bindingof 18 to the ground state was confirmed by dramatically slowed tetramerdissociation in 6 M urea and sluggish amyloidogenicity at pH 4.4. OH-PCB18 (3.6 μM) is believed to be an impressive amyloid inhibitor because itis an excellent kinetic stabilizer of tetrameric TTR, i.e. it prevents ⅔of a 3.6 μM TTR sample from being amyloidogenic at pH 4.4 because TTR•18and TTR•18₂ are incompetent to form amyloid, the remainder of TTR (1.18μM) forms amyloid very inefficiently because of its low concentration.The dissociation rates of the best OH-PCB inhibitors may also be slowerthan expected because of TTR structural annealing around the OH-PCB, butthis has not yet been evaluated as carefully as required. At a minimum,these compounds provide guidance for the synthesis of exceptionalinhibitors, or may themselves prove useful as inhibitors depending ontheir toxicity profile.

The structural information on TTR bound to OH-PCBs 12, 16, 17 and 18reveal that these biaryls generally bind along the crystallographictwo-fold symmetry axis. The dihedral angle between the two rings rangesfrom ˜40–60°, allowing the halogen binding pockets (HBPs) on twoneighboring subunits to be engaged simultaneously, leading tostabilization of the tetrameric quaternary structural interface.Hydroxylated PCB 18 has optimal structural complimentarily with TTR asits chlorines are able to bind to HBPs 1 and 1′ as well as 3 and 3′simultaneously. This is not the case with 16 and 17, which requireconsideration of both symmetry equivalent binding modes in order toextend chlorines into HBPS 1, 1′, 3 and 3′.

The orientation of the phenolic ring into the inner binding pocketappears to play a important role in that it enables a water mediatedhydrogen bonding network to form between it and neighboring TTR subunitsthat presumably further stabilizes the native quaternary structure ofTTR. A H-bonding network involving the three staggered conformations ofSer-117, the phenolic group of the inhibitor and the two conserved watermolecules creates an electrostatic network that interconnects the twosubunits that form the PCB binding site. In all three structures, Thr119also occupies multiple rotomer conformations. In contrast, this networkof electrostatic interactions is absent in the 12₂•TTR complex in whichthe hydroxyl substituted phenyl ring is oriented in the outer bindingpocket and wherein Ser 117 and Thr 119 adopt apo side chainconformations.

The toxicity of OH-PCBs is not well established in the literature. In avariety of in vitro and animal studies, OH-PCBs appear to be eithermildly estrogenic or anti-estrogenic. Other toxicity mechanisms havebeen suggested and there are also reports of decreased thyroid hormonelevels in animals exposed to these compounds. The suggestion that OH-PCBbinding to TTR lowers T4 levels and that lowered T4 levels reflectssmall molecule TTR binding is difficult to directly support. Sinceroughly half of T4 is carried by albumin, the displacement of T4 fromthe albumin binding sites seems more likely to be the cause the loweredT4 levels in individuals exposed to PCBs. Thyroid binding globulin hasthe highest affinity for thyroxine and is a main carrier in humans, butit is not present in many lower mammals, including rats and mice wheremany of the toxicological profiles of these compounds have been studied.Thus, in these species it is more likely that compounds binding to TTRwill have an effect on the overall binding and transport of T4. Datashowing binding of PCBs to TBG suggest little interaction, with theexception of one or two weakly binding compounds. Therefore, the effectof OH-PCBs on human thyroid levels should be minimal unless they bind toalbumin. There are also reports that these compounds may be interferingwith thyroid hormone activation or increasing the rate of sulfation, andtherefore inactivation, of T4. OH-PCBs could also bind to other thyroidhormone targets including thyroid hormone receptors, which seemsreasonable given the structural analogy with T4.

It is clear that little is established regarding hydroxylated PCBtoxicity, especially in humans. The toxicology in rodents is expected tobe more severe owing to TTR's role as the primary thyroid hormonetransporter. What is clear is that hydroxylated PCBs exhibit excellentactivity as inhibitors of transthyretin fibril formation, suggestingthat this class of compounds has the potential to be useful for theinhibition of amyloid fibril formation.

Materials and Methods

Transthyretin Antibody Purification and Conjugation to Sepharose

Antibodies were produced, purified and coupled to Sepharose. The resinwas stored as a 1:1 slurry in TSA (10 mM Tris, pH 8.0/140 mM NaCl/0.025%NaN₃). In addition, quenched Sepharose was prepared by coupling 200 mMTris, pH 8.0 to the resin instead of the antibody.

Human Plasma Preparation

Whole blood was drawn from healthy volunteers at the Scripps GeneralClinical Research Center's Normal Blood-Drawing Program and transferredto 50 mL conical tubes. The tubes were centrifuged at 3000 RPM (1730×g)in a Sorvall RT7 benchtop centrifuge equipped with a swinging bucketrotor for 10 min at 25° C. The plasma supernatant was removed andcentrifuged again at 3000 RPM for 10 min to remove the remaining cells.Sodium azide was added to give a 0.05% solution. The plasma was storedat 4° C. until use

Immunoprecipitation of Transthyretin and Bound PCBs

A 2 mL eppendorf tube was filled with 1.5 mL of human blood plasma and7.5 μL of a 2.16 mM DMSO solution of the PCB under evaluation. Thissolution was incubated at 37° C. for 24 h. A 1:1 resin/TSA slurry (187μL) of quenched Sepharose was added to the solution and gently rocked at4° C. for 1 h. The solution was centrifuged (16,000×g) and thesupernatant divided into 3 aliquots of 400 μL each. These were eachadded to 200 μL of a 1:1 resin/TSA slurry of the anti-transthyretinantibody-conjugated Sepharose and slowly rocked at 4° C. for 20 min. Thesamples were centrifuged (16,000×g) and the supernatant removed. Theresin was washed with 1 mL TSA/0.05% Saponin (Acros) (3×10 min) at 4°C., and additionally with 1 mL TSA (2×10 min) at 4° C. The samples werecentrifuged (16,000×g), the final wash removed, and 155 μL of 100 mMtriethylamine, pH 11.5 was added to elute the TTR and bound smallmolecules from the antibodies. Following gentle rocking at 4° C. for 30min, the samples were centrifuged (16,000×g) and 145 μL of thesupernatant, containing TTR and inhibitor, was removed.

HPLC Analysis and Quantification of Transthyretin and Bound PCBs

The supernatant elution samples from the TTR antibody beads (145 μL)were loaded onto a Waters 71P autosampler. A 135 μL injection of eachsample was separated on a Keystone 3 cm C18 reverse phase columnutilizing a 40–100% B gradient over 8 min (A: 94.8% H₂O/5%acetonitrile/0.2% TFA; B: 94.8% acetonitrile/5% H₂O/0.2% TFA),controlled by a Waters 600E multisolvent delivery system. Detection wasaccomplished at 280 nm with a Waters 486 tunable absorbance detector,and the peaks were integrated to give the area of both TTR and the smallmolecule. In order to determine the quantity of each species, knownamounts of tetrameric TTR or PCB were injected onto the HPLC. The peakswere integrated to create calibration curves from linear regressions ofthe data using Kaleidagraph (Synergy Software). The calibration curveswere used to determine the number of moles of each species present inthe plasma samples. The ratio of small molecule to protein wascalculated to yield the stoichiometry of small molecule bound to TTR inplasma.

Transthyretin Amyloid Fibril Formation Assay

The compounds were dissolved in DMSO at a concentration of 720 μM. FiveμL of a solution of the compound being evaluated was added to 0.5 mL ofa 7.2 μM TTR solution in 10 mM phosphate pH 7.6, 100 mM KCl, 1 mM EDTAbuffer, allowing the compound to incubate with TTR for 30 min. 495 μL of0.2 mM acetate pH 4.2, 100 mM KCl, 1 mM EDTA was added, to yield finalprotein and inhibitor concentrations of 3.6 μM each and a pH of 4.4. Themixture was then incubated at 37° C. for 72 h, after which the tubeswere vortexed for 3 sec and the optical density was measured at 400 nm.The extent of fibril formation was determined by normalizing eachoptical density by that of TTR without inhibitor, defined to be 100%fibril formation. Control solutions of each compound in the absence ofTTR were also tested and none absorbed appreciably at 400 nm.

Isothermal Titration Calorimetry of PCB 18 and TTR

A 25 μM solution of compound 18 (in 10 mM phosphate pH 7.6, 100 mM KCl,1 mM EDTA, 8% DMSO) was titrated into a 1.2 μM solution of TTR in anidentical buffer using a Microcal MCS Isothermal Titration Calorimeter(Microcal, Northampton, Mass.). An initial injection of 2 μL wasfollowed by 25 injections of 10 μL at 25° C. The thermogram wasintegrated and a blank was subtracted to yield a binding isotherm thatfit best to a model of two identical binding sites using the ITC dataanalysis package in ORIGIN 5.0 (Microcal).

Crystallization and X-Ray Data Collection

Crystals of recombinant TTR were obtained from protein solutions at 5mg/ml (in 100 mM KCl, 100 mM phosphate, pH 7.4, 1 M ammonium sulfate)equilibrated against 2 M ammonium sulfate in hanging drop experiments.The TTR•ligand complexes were prepared from crystals soaked for 2 weekswith a 10-fold molar excess of the ligand to ensure full saturation ofboth binding sites. 1:1 acetone:water solution was used as a soakingagent. A DIP2030b imaging plate system (MAC Science, Yokohama, Japan)coupled to a RU200 rotating anode X-ray generator was used for datacollection. The crystals were placed in paratone oil as acryo-protectant and cooled to 120 K for the diffraction experiments.Crystals of all TTR•ligand complexes are isomorphous with the apocrystal form containing unit cell dimensions a=43 Å, b=86 Å and c=65 Å.They belong to the space group P2₁2₁2 and contain half of thehomotetramer in the asymmetric unit. Data were reduced with DENZO andSCALEPACK.

Structure Determination and Refinement

The protein atomic coordinates for TTR from the Protein Data Bank(accession number 1BMZ) were used as a starting model for the refinementof native TTR and the TTR-ligand complexes by molecular dynamics andenergy minimization using the program CNS. Maps were calculated fromdiffraction data collected on TTR crystals either soaked with PCBs orcocrystalized simultaneously. For the complexes of TTR with the PCBs,the resulting maps revealed approximate positions of the ligand in bothbinding pockets of the TTR tetramer, with peak heights of above 5–9r.m.s. In order to further improve the small molecule electron densityand remove the model bias, the model was subjected to several cycles ofthe warp/shake protocol, which resulted in noticeable improvement in themap, especially around the inhibitor. Subsequent model fitting was doneusing these maps and the ligand molecule was placed into the density. Inall three cases the minimum-energy conformation of the inhibitorcalculated by the program InsightII (Accelrys) was in good agreementwith the map. Because of the two-fold crystallographic symmetry axisalong the binding channel, a statistical disorder model must be applied,giving rise to two ligand binding modes in each of the two binding sitesof tetrameric TTR. Water molecules were added based upon the unbiasedelectron density map. Because of the lack of interpretable electrondensities in the final map, the nine N-terminal and three C-terminalresidues were not included in the final model.

Example 5 Benzoxazoles as Transthyretin Amyloid Fibril Inhibitors

Transthyretin's two thyroxine binding sites are created by itsquaternary structural interface. The tetramer can be stabilized by smallmolecule binding to these sites, potentially providing a means to treatTTR amyloid disease with small molecule drugs. Many families ofcompounds have been discovered whose binding stabilizes the tetramericground state to a degree proportional to the small molecule dissociationconstants K_(d1) and K_(d2). This also effectively increases thedissociative activation barrier and inhibits amyloidosis by kineticstabilization. Such inhibitors are typically composed of two aromaticrings, with one ring bearing halogen substituents and the other bearinghydrophilic substituents. Benzoxazoles substituted with a carboxylicacid at C(4)–C(7) and a halogenated phenyl ring at C(2) also appeared tocomplement the TTR thyroxine binding site. A small library of thesecompounds was therefore prepared by dehydrocyclization of N-acylamino-hydroxybenzoic acids as illustrated in Scheme 1.

Reagents: (a) ArCOCl, THF, pyridine (Ar=Phenyl, 3,5-Difluorophenyl,2,6-Difluorophenyl, 3,5-Dichlorophenyl, 2,6-Dichlorophenyl,2-(Trifluoromethyl)phenyl, and 3-(Trifluoromethyl)phenyl); (b) TsOH•H₂O,refluxing xylenes; (c) TMSCHN₂, benzene, MeOH; (d) LiOH, THF, MeOH, H₂O(8–27% yield over 4 steps).

The benzoxazoles were evaluated using a series of analyses of increasingstringency. WT TTR (3.6 μM) was incubated for 30 min (pH 7, 37° C.) witha test compound (7.2 μM). Since at least one molecule of the testcompound must bind to each molecule of TTR tetramer to be able tostabilize it, a test compound concentration of 7.2 μM is only twice theminimum effective concentration. The pH was then adjusted to 4.4, theoptimal pH for fibrilization. The amount of amyloid formed after 72 h(37° C.) in the presence of the test compound was determined byturbidity at 400 nm and is expressed as % fibril formation (ff), 100%being the amount formed by TTR alone. Of the 28 compounds tested, 11reduced fibril formation to negligible levels (ff<10%; FIG. 7).

The 11 most active compounds were then evaluated for their ability tobind selectively to TTR over all other proteins in blood. Human bloodplasma (TTR conc. 3.6–5.4 μM) was incubated for 24 h with the testcompound (10.8 μM) at 37° C. The TTR and any bound inhibitor wereimmunoprecipitated using a sepharose-bound polyclonal TTR antibody. TheTTR with or without inhibitor bound was liberated from the resin at highpH, and the inhibitor:TTR stoichiometry was ascertained by HPLC analysis(FIG. 8). Benzoxazoles with carboxylic acids in the 5- or 6-position,and 2,6-dichlorophenyl (13, 20) or 2-trifluoromethylphenyl (11, 18)substituents at the 2-position displayed the highest bindingstoichiometries. In particular, 20 exhibited excellent inhibitoryactivity and binding selectivity. Hence, its mechanism of action wascharacterized further.

To confirm that 20 inhibits TTR fibril formation by binding strongly tothe tetramer, isothermal titration calorimetry (ITC) and sedimentationvelocity experiments were conducted with wt TTR. ITC showed that twoequivalents of 20 bind with average dissociation constants ofK_(d1)=K_(d2)=55 (±10) nM under physiological conditions. These arecomparable to the dissociation constants of many other highlyefficacious TTR amyloidogenesis inhibitors. For the sedimentationvelocity experiments, TTR (3.6 μM) was incubated with 20 (3.6 μM, 7.2μM, 36 μM) under optimal fibrilization conditions (72 h, pH 4.4, 37°C.). The tetramer (55 kDa) was the only detectable species in solutionwith 20 at 7.2 or 36 μM. Some large aggregates formed with 20 at 3.6 μM,but the TTR remaining in solution was tetrameric.

T119M subunit inclusion and small molecule binding both prevent TTRamyloid formation by raising the activation barrier for tetramerdissociation. An inhibitor's ability to do this is most rigorouslytested by measuring its efficacy at slowing tetramer dissociation in 6 Murea, a severe denaturation stress. Thus, the rates of TTR tetramerdissociation in 6 M urea in the presence and absence of 20, 21 or 27were compared (FIG. 9). TTR (1.8 μM) was completely denatured after 168h in 6 M urea. In contrast, 20 at 3.6 μM prevented tetramer dissociationfor at least 168 h (>3× the half-life of TTR in human plasma). With anequimolar amount of 20, only 27% of TTR denatured in 168 h. Compound 27(3.6 μM) was much less able to prevent tetramer dissociation (90%unfolding after 168 h), even though it was active in the fibrilformation assay. Compound 21 did not hinder the dissociation of TTR atall. These results show that inhibitor binding to TTR is necessary butnot sufficient to kinetically stabilize the TTR tetramer under stronglydenaturing conditions; it is also important that the dissociationconstants be very low (or that the off rates be very slow). Also, thedisplay of functional groups on 20 is apparently optimal for stabilizingthe TTR tetramer; moving the carboxylic acid from C(6) to C(7), as in27, or removing the chlorines, as in 21, severely diminishes itsactivity.

The role of the substituents in 20 is evident from its co-crystalstructure with TTR (FIG. 10). Compound 20 orients its two chlorine atomsnear halogen binding pockets 2 and 2′ (so-called because they areoccupied by iodines when thyroxine binds to TTR). The 2,6 substitutionpattern on the phenyl ring forces the benzoxazole and phenyl rings outof planarity, optimally positioning the carboxylic acid on thebenzoxazole to hydrogen bond to the ε-NH₃ ⁺ groups of Lys 15/15′.Hydrophobic interactions between the aromatic rings of 20 and the sidechains of Leu 17, Leu 110, Ser 117, and Val 121 contribute additionalbinding energy.

Methods

The general procedure for benzoxazole synthesis and characterization ofthe products (¹H- and ¹³C-NMR and high resolution mass spectra) aredetailed below.

Analytical Ultracentrifugation

The quaternary structure of TTR in the presence of 20 was observed usingsedimentation velocity analytical ultracentrifugation. The samples wereincubated with 20 at 3.6, 7.2 or 36 μM for 72 h. The data were collectedon a temperature-controlled Beckman XL-I analytical ultracentrifuge(equipped with an An60Ti rotor and photoelectric scanner). A doublesector cell, equipped with a 12 mm Epon centerpiece and sapphirewindows, was loaded with 400–420 μL of sample using a syringe. Data werecollected at rotor speeds of 3000 and 50000 rpm in continuous mode at25° C., with a step size of 0.005 cm employing an average of 1 scan perpoint. Detection was carried out at 280 nm. The data were subjected totime-derivative analysis using the program DCDT+ developed by Philo(Philo, 2000; Stafford, 1992). The analysis showed that the distributionof species in solution represented by a range of s values. Thisdistribution was then fitted to various models in order to determine thesedimentation and diffusion coefficients for species in the system. Themolecular weight of each species was determined by methods reportedpreviously (Petrassi, et al 2000). The s values found for TTR showedthat it remained tetrameric in the presence of 7.2 and 36 μM of 20,while at 3.6 μM the TTR remaining in solution was tetrameric despite theformation of some aggregate.

Crystallization and X-Ray Data Collection

Crystals of wt TTR were obtained from protein solutions at 12 mg/mL (in100 mM KCl, 1 mM EDTA, 10 mM Sodium phosphate, and pH 7.0, 0.35 Mammonium sulfate) equilibrated against 2 M ammonium sulfate in hangingdrop experiments. The TTR-20 complex was prepared from crystals soakedfor 3 wk with a 10 fold molar excess of the ligand to ensure fullsaturation of both binding sites. The ligand-soaked crystal diffractedup to 1.55 Å on a Quantum-4 detector at the monochromatic high energysource of 14-BM-C, BIOCARS, Advanced Photon Source (Argonne NationalLaboratory). The crystals were soaked in paratone oil and flash-cooledto 100 K for the diffraction experiments. Crystals of the TTR-20 complexare isomorphous with the apo crystal form with unit cell dimensionsa=43.1 Å, b=84.7 Å, and c=64.7 Å, space group P2₁2₁2 with two TTRsubunits in the asymmetric unit. Data were reduced with DENZO andSCALEPACK of the HKL2000 suite. (Otwinowski, 1997)

Structure Determination and Refinement

The protein atomic coordinates for TTR from the Protein Data Bank(accession number 1BMZ) were used as a starting model for the molecularreplacement searches. The refinement of the TTR-20 complex structure wascarried out using molecular dynamics and the energy minimizationprotocols of CNS. The resulting difference Fourier maps revealed bindingof the ligand in both binding pockets of the TTR tetramer. Using thesemaps, the ligand could be unambiguously placed into the density and wasincluded in the crystallographic refinement. The minimum energyconformation of the inhibitor calculated by the program Insight II(Accelrys Inc.) was used as the initial model for the crystallographicrefinement. Because the 2-fold crystallographic symmetry axis is alongthe binding channel, a statistical disorder model had to be applied,giving rise to two ligand binding modes per TTR binding pocket. Afterseveral cycles of simulated annealing and subsequent positional andtemperature factor refinement, water molecules were placed into thedifference Fourier maps. The final cycle of map fitting was done usingthe unbiased weighted electron density map calculated by the shake n'warp bias removal protocol. The symmetry related binding conformationsof the ligand were in good agreement with the unbiased annealed omitmaps as well as the shake n' warp unbiased weighted maps phased in theabsence of the inhibitor. Because of the lack of interpretable electrondensities in the final map, the nine N-terminal and three C-terminalresidues were not included in the final model. A summary of thecrystallographic analysis is presented in Table 6.

TABLE 6 Statistics for X-ray Crystal Structure Completeness (%)(overall/outer shell) 86/90 R_(sym) (Overall/outer shell) 0.05/0.33Refinement statistics Resolution (Å) 33.02–1.55 R-factor/R-free (%)21.1/24.3 Rmsd bond length (Å) 0.03 Rmsd bond angles (°) 2.5 Otherstatistics Crystal dimensions (mm) 0.3 × 0.2 × 0.15 Crystal systemOrthorhombic Unit cell dimensions (a, b, c in Å) 43.1, 84.7, 64.7 Unitcell volume (Å³) 236123 Maximum resolution (Å) 1.54 Scan mode PhiTemperature of measurement 100 K Number of independent reflections 30705Method of structure solution Molecular replacement by EPMR (Kissinger,1999) Refinement against F_(obs) Refinement target Maximum likelihoodProgram used for refinement CNS-Solve (Brunger 1998) Database ProteinData Bank

Benzoxazole Synthesis—General Methods

Unless stated otherwise, all reactions were carried out in oven-driedglassware under a dry argon atmosphere using a FirstMate OrganicSynthesizer (Argonaut Technologies). All solvents (anhydrous) andreagents were purchased from Aldrich and used without furtherpurification. ¹H NMR spectra were measured at 500 MHz on a BrukerDRX-500 spectrometer or at 600 MHz on a Bruker DRX-600 spectrometer, andwere referenced to internal CHD₂-S(O)—CD₃ (2.49 ppm). ¹³C spectra wereperformed at 125 MHz on a Bruker DRX-500 or at 150 MHz on a BrukerDRX-600 instrument and were referenced to (CD₃)₂SO (39.5 ppm).Thin-layer chromatographic analyses were performed on Glass-backedthin-layer analytical plates (Kieselgel 60 F₂₅₄, 0.25 mm, EM Science no.5715-7). Visualization was accomplished using UV absorbance or 10%phosphomolybdic acid in ethanol. Chromatography was performed on achromatotron (Harrison Research, Model 7924T, 2 mm plate) or on apreparative silica gel plate (Kieselgel 60 F₂₅₄, 1 mm, EM Science no.13895-7).

General Procedure for Benzoxazole Synthesis

A mixture of amino hydroxybenzoic acid (0.2 mmol) in THF (3 mL) wassequentially treated with pyridine (500 μl, 0.6 mmol) and the desiredacid chloride (0.2 mmol). The reaction mixture was stirred at ambienttemperature for 10 h, refluxed for 1 h, concentrated in vacuo and usedin the next step without purification.

p-Toluenesulfonic acid monohydrate (380.4 mg, 2.0 mmol) was added to thecrude reaction mixture in xylenes (5 mL) and the resulting mixture wasstirred at reflux overnight. After 12 h, the reaction was cooled toambient temperature, quenched with NaOH (2 mL, 1 N) and the phases wereseparated. The aqueous layer was acidified with HCl (1 N) to pH 2 andextracted with EtOAc (4×3 mL). The combined organic layers were driedover MgSO₄, filtered and concentrated in vacuo. The resulting residuewas dissolved in a mixture of MeOH:Benzene (2 mL; 1:4), treated withTMS-CHN₂ (200 μL of 2.0 M solution in hexanes, 0.4 mmol) at 25° C. andthe reaction progress was monitored by TLC (usually complete after 0.5h). The reaction mixture was concentrated in vacuo, and the residue waschromatographed (10 to 25% EtOAc/hexanes gradient) to afford the desiredbenzoxazole methyl ester.

The benzoxazole methyl ester was dissolved in a mixture of THF:MeOH:H₂O(3:1:1, 0.07 M) and treated with LiOH.H₂O (4 equiv). The reaction wasstirred at ambient temperature and monitored by TLC. Upon completion,the mixture was acidified to pH 2 with 1 N HCl and extracted with EtOAc(4×). The combined organic layers were dried over MgSO₄, filtered andconcentrated. The residue was purified by preparative thin layerchromatography (4.9% MeOH, 95% CH₂Cl₂, 0.1% HOAc) to give the product asa white solid.

4-Carboxy-2-(3,5-difluorophenyl)-benzoxazole (1). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford1 as a white solid (7.0 mg, 13%). Data for 1: ¹H NMR (500 MHz, DMSO-d₆)δ 13.70–12.50 (br. s, 1H, CO₂H), 8.04 (AMX, 1H, J=8.1 Hz, Ar), 7.94(AMX, 1H, J=7.3 Hz, Ar), 7.84 (br. d, 2H, J=5.6 Hz, Ar), 7.62–7.58 (m,1H, Ar), 7.56 (AMX, 1H, J=7.3, 8.1 Hz, Ar); ¹³C NMR (125 MHz, DMSO-d₆) δ165.8, 162.7 (d, J=248 Hz), 162.6 (d, J=248 Hz), 161.1, 151.0, 140.3,129.3, 127.0, 125.8, 123.6, 115.2, 110.8 (d, J=28 Hz), 107.8 (t, J=26Hz); HRMS (MALDI-FTMS) calcd. for C₁₄H₇F₂NO₃ (MH⁺) 276.0467, found276.0463.

4-Carboxy-2-(2,6-difluorophenyl)-benzoxazole (2). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford2 as a white solid (8.2 mg, 15%). Data for 2: ¹H NMR (500 MHz, DMSO-d₆)δ 13.00 (br. s, 1H, CO₂H), 8.06 (AMX, 1H, J=8.1 Hz, Ar), 7.94 (AMX, 1H,J=7.6 Hz, Ar), 7.80–7.74 (m, 1H, Ar), 7.57 (AMX, 1H, J=7.6, 8.1 Hz, Ar),7.40–7.38 (m, 2H, Ar); ¹³C NMR (125 MHz, DMSO-d₆) □166.1, 160.4 (d,J=256 Hz), 160.3 (d, J=256 Hz), 154.9, 150.6, 139.6, 134.7 (t, J=10 Hz),126.8, 125.8, 114.8, 112.8 (d, J=22 Hz), 105.2 (t, J=16 Hz); HRMS(MALDI-FTMS) calcd. for C₁₄H₇F₂NO₃ (MH⁺) 276.0467, found 276.0461.

4-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole (3). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford3 as a white solid (9.5 mg, 15%). Data for 3: ¹H NMR (500 MHz, DMSO-d₆)δ 13.70–12.80 (br. s, 1H, CO₂H), 8.50 (ABX, 1H, J=7.8 Hz, Ar), 8.43 (s,1H, Ar), 8.06 (AMX, 1H, J=8.1 Hz, Ar), 8.03 (ABX, 1H, J=8.1 Hz, Ar),7.94 (AMX, 1H, J=7.8 Hz, Ar), 7.88 (ABX, 1H, J=7.8 Hz, Ar), 7.54 (AMX,1H, J=8.1 Hz, Ar); ¹³C NMR (125 MHz, DMSO-d₆) δ 165.8, 161.9, 151.0,140.6, 131.4, 130.8, 130.0 (q, J=33 Hz), 128.7 (d, J=4 Hz), 127.2,127.0, 125.5, 123.8, 123.7 (q, J=273 Hz), 123.2, 115.2; HRMS(MALDI-FTMS) calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0535.

4-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole (4). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford4 as a white solid (15.2 mg, 25%). Data for 4: ¹H NMR (600 MHz, DMSO-d₆)δ 13.15 (br. s, 1H, CO₂H), 8.18 (d, 1H, J=7.6 Hz, Ar), 8.06 (AMX, 1H,J=0.9, 8.2 Hz, Ar), 8.02 (d, 1H, J=7.9 Hz, Ar), 7.96 (AMX, 1H, J=0.9,7.9 Hz, Ar), 7.94–7.87 (m, 2H, Ar), 7.58 (AMX, 1H, J=8.2 Hz, Ar): ¹³CNMR (150 MHz, DMSO-d₆) δ 165.8, 161.6, 151.2, 140.0, 133.0, 132.6,132.3, 127.6 (q, J=32 Hz), 127.2 (q, J=6 Hz), 127.0, 125.6, 124.9,123.5, 123.4 (q, J=273 Hz), 115.2; HRMS (MALDI-FTMS) calcd. forC₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0531.

4-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole (5). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford5 as a white solid (8.0 mg, 13%). Data for 5: ¹H NMR (600 MHz, DMSO-d₆)δ 13.60–12.60 (br. s, 1H, CO₂H), 8.16 (A₂ M, 2H, J=2.0 Hz, Ar), 8.05(AMX, J=0.9, 8.2 Hz, Ar), 7.96 (A₂ M, 1H, J=2.0 Hz, Ar), 7.94 (AMX, 1H,J=0.9, 7.6 Hz, Ar), 7.56 (AMX, 1H, J=7.9 Hz, Ar); ¹³C NMR (150 MHz,DMSO-d₆) δ 165.8, 160.8, 151.0, 140.4, 135.2, 131.5, 129.4, 127.0,126.3, 125.9, 125.8, 123.6, 115.2; HRMS (MALDI-FTMS) calcd. forC₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9876.

4-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole (6). Prepared from3-hydroxyanthranilic acid according to the general procedure, to afford6 as a white solid (5.2 mg, 8%). Data for 6: ¹H NMR (600 MHz, DMSO-d₆) δ13.80–12.50 (br. s, 1H, CO₂H), 8.07 (AMX, 1H, J=8.2 Hz, Ar), 7.95 (AMX,1H, J=7.9 Hz, Ar), 7.77–7.71 (m, 3H, Ar), 7.59 (AMX, 1H, J=7.9, 8.2 Hz,Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 165.8, 158.2, 150.8, 139.3, 134.8,134.0, 128.7, 126.8, 126.7, 125.9, 122.4; HRMS (MALDI-FTMS) calcd. forC₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9880.

4-Carboxy-2-phenyl-benzoxazole (7). Prepared from 3-hydroxyanthranilicacid according to the general procedure, to afford 7 as a white solid(10.2 mg, 21%). Data for 7: ¹H NMR (600 MHz, DMSO-d₆) δ 13.50–12.60 (br.s, 1H, CO₂H), 8.24–8.22 (m, 2H, Ar), 8.03 (AMX, 1H, J=0.9, 8.2 Hz, Ar),7.91 (AMX, 1H, J=0.9, 7.9 Hz, Ar), 7.68 –7.61 (m, 3H, Ar), 7.51 (AMX,1H, J=7.9, 8.2 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 166.0, 163.4,151.0, 140.8, 132.4, 129.4, 127.6, 126.7, 126.1, 125.0, 123.0, 115.0;HRMS (MALDI-FTMS) calcd. for C₁₄H₉NO₃ (MH⁺) 240.0655, found 240.0656.

5-Carboxy-2-(3,5-difluorophenyl)-benzoxazole (8). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 8 as a white solid (10.2 mg, 19%). Data for 8: ¹H NMR (600 MHz,DMSO-d₆) δ 13.60–12.80 (br. s, 1H, CO₂H), 8.32 (ABM, 1H, J=1.5 Hz, Ar),8.07 (ABM, 1H, J=1.5, 8.5 Hz, Ar), 7.90 (ABM, 1H, J=8.5 Hz, Ar),7.86–7.85 (m, 2H, Ar), 7.60 (tt, 1H, J=2.4, 9.2 Hz, Ar); ¹³C NMR (150MHz, DMSO-d₆) δ 166.8, 162.8 (d, J=248 Hz), 162.7 (d, J=248 Hz), 161.5,153.0, 141.2, 129.1 (t, J=11 Hz), 128.2, 127.7, 121.4, 111.2, 110.8 (d,J=23 Hz), 110.7 (d, J=22 Hz), 107.8 (t, J=26 Hz); HRMS (MALDI-FTMS)calcd. for C₁₄H₇F₂NO₃ (MH⁺) 276.0467, found 276.0469.

5-Carboxy-2-(2,6-difluorophenyl)-benzoxazole (9). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 9 as a white solid (6.8 mg, 12%). Data for 9: ¹H NMR (600 MHz,DMSO-d₆) δ 13.50–12.80 (br. s, 1H, CO₂H), 8.39 (ABM, 1H, J=0.7, 1.6 Hz,Ar), 8.10 (ABM, 1H, J=1.6, 8.7 Hz, Ar), 7.95 (ABM, 1H, J=0.7, 8.7 Hz,Ar), 7.77 (m, 1H, Ar), 7.40 (t, 2H, J=8.8 Hz, Ar); 3C NMR (150 MHz,DMSO-d₆) δ 166.8, 160.4 (d, J=257 Hz), 160.3 (d, J=257 Hz), 155.4,152.6, 140.8, 134.8 (t, J=11 Hz), 128.2, 127.7, 121.6, 113.0 (d, J=22Hz), 112.9 (d, J=22 Hz), 111.2, 104.9; HRMS (MALDI-FTMS) calcd. forC₁₄H₇F₂NO₃ (MH⁺) 276.0467, found 276.0467.

5-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole (10). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 10 as a white solid (6.7 mg, 11%). Data for 10: ¹H NMR (500 MHz,DMSO-d₆) δ 13.30–12.80 (br. s, 1H, CO₂H), 8.51 (ABX, 1H, J=7.8 Hz, Ar),8.45 (s, 1H, Ar), 8.35 (ABM, 1H, J=1.7 Hz, Ar), 8.08 (ABM, 1H, J=1.7,8.6 Hz, Ar), 8.04 (ABX, 1H, J=7.8 Hz, Ar), 7.93 (ABM, 1H, J=8.6 Hz, Ar),7.89 (ABX, 1H, J=7.8 Hz, Ar); ¹³C NMR (125 MHz, DMSO-d₆) δ 166.8, 162.2,153.1, 141.4, 131.4, 130.9, 130.1 (q, J=33 Hz), 128.8, 128.2, 127.5,127.1, 123.8 (q, J=4 Hz), 123.7 (q, J=273 Hz), 121.3, 111.2; HRMS(MALDI-FTMS) calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0530.

5-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole (11). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 11 as a white solid (10.3 mg, 17%). Data for 11: ¹H NMR (600 MHz,DMSO-d₆) δ 13.19 (br. s, 1H, CO₂H), 8.38 (m, 1H, Ar), 8.19 (d, 1H, J=7.6Hz, Ar), 8.09 (dd, 1H, J=1.8, 8.5 Hz, Ar), 8.03 (d, 1H, J=7.9 Hz, Ar),7.94–7.88 (m, 3H, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 166.8, 161.6, 153.2,141.1, 133.1, 132.5, 132.4, 128.2, 127.6, 127.5 (q, J=32 Hz), 127.2 (q,J=6 Hz), 124.7, 123.4 (q, J=274 Hz), 121.6, 111.2; HRMS (MALDI-FTMS)calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0531.

5-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole (12). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 12 as a white solid (7.3 mg, 12%). Data for 12: ¹H NMR (600 MHz,DMSO-d₆) δ 13.14 (br. s, 1H, CO₂H), 8.33 (AMX, 1H, J=0.6, 1.8 Hz, Ar),8.16 (AM, 2H, J=1.8 Hz, Ar), 8.08 (AMX, 1H, J=1.8, 8.5 Hz, Ar), 7.95(AM, 1H, J=1.8 Hz, Ar), 7.91 (AMX, 1H, J=0.6, 8.5 Hz, Ar); ¹³C NMR (150MHz, DMSO-d₆) δ 166.7, 161.1, 153.0, 141.3, 135.2, 131.6, 129.2, 128.2,127.7, 125.9, 121.4, 111.3; HRMS (MALDI-FTMS) calcd. for C₁₄H₇Cl₂NO₃(MH⁺) 307.9876, found 307.9879.

5-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole (13). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 13 as a white solid (10.8 mg, 18%). Data for 13: ¹H NMR (600 MHz,DMSO-d₆) δ 13.08 (br. s, 1H, CO₂H), 8.43 (AMX, 1H, J=0.6, 1.8 Hz, Ar),8.13 (AMX, 1H, J=1.8, 8.5 Hz, Ar), 7.98 (AMX, 1H, J=0.6, 8.5 Hz, Ar),7.77–7.72 (m, 3H, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 166.7, 158.6, 152.8,140.4, 134.8, 134.2, 128.8, 128.4, 127.8, 126.2, 121.8, 111.5; HRMS(MALDI-FTMS) calcd. for C₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9879.

5-Carboxy-2-phenyl-benzoxazole (14). Prepared from3-amino-4-hydroxybenzoic acid according to the general procedure, toafford 14 as a white solid (11.5 mg, 24%). Data for 14: ¹H NMR (600 MHz,DMSO-d₆) δ 13.12 (br. s, 1H, CO₂H), 8.30 (ABX, 1H, J=1.8 Hz, Ar), 8.20(dt, 2H, J=1.5, 6.7 Hz, Ar), 8.03 (ABX, 1H, J=1.8, 8.5 Hz, Ar), 7.87(ABX, 1H, J=8.5 Hz, Ar), 7.67–7.60 (m, 3H, Ar); ¹³C NMR (150 MHz,DMSO-d₆) δ 166.9, 163. 153.0, 141.6, 132.4, 129.4, 127.9, 127.5, 127.0,126.0, 121.0, 111.0; HRMS (MALDI-FTMS) calcd. for C₁₄H₉NO₃ (MH⁺)240.0655, found 240.0656.

6-Carboxy-2-(3,5-difluorophenyl)-benzoxazole (15). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 15 as a white solid (10.3 mg, 19%). Data for 15: ¹H NMR (600 MHz,DMSO-d₆) δ 13.22 (br. s, 1H, CO₂H), 8.20 (ABM, 1H, J=1.5 Hz, Ar), 7.98(ABM, 1H, J=1.5, 8.2 Hz, Ar), 7.86 (ABM, 1H, J=8.2 Hz, Ar), 7.79–7.78(m, 2H, Ar), 7.57 (tt, 1H, J=2.4, 9.4 Hz, Ar); ¹³C NMR (150 MHz,DMSO-d₆) δ 166.7, 162.7 (d, J=248 Hz), 162.6 (d, J=248 Hz), 162.4,150.0, 144.7, 129.0 (t, J=11 Hz), 128.7, 126.5, 120.0, 112.1, 110.9 (d,J=23 Hz), 110.8 (d, J=22 Hz), 108.0 (t, J=26 Hz); HRMS (MALDI-FTMS)calcd. for C₁₄H₇F₂NO₃ (MH⁺) 276.0467, found 276.0468.

6-Carboxy-2-(2,6-difluorophenyl)-benzoxazole (16). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 16 as a white solid (8.5 mg, 15%). Data for 16: ¹H NMR (600 MHz,DMSO-d₆) δ 13.25 (br. s, 1H, CO₂H), 8.30 (ABM, 1H, J=0.6, 1.5 Hz, Ar),8.04 (ABM, 1H, J=1.5, 8.2 Hz, Ar), 7.96 (ABM, 1H, J=0.6, 8.2 Hz, Ar),7.76 (m, 1H, Ar), 7.39 (t, 2H, J=8.8 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆)δ 166.7, 160.4 (d, J=257 Hz), 160.3 (d, J=257 Hz), 156.6, 149.7, 144.2,134.9 (t, J=11 Hz), 128.8, 126.4, 120.1, 113.1, 112.9, 112.2 (d, J=5Hz), 105.0 (t, J=16 Hz); HRMS (MALDI-FTMS) calcd. for C₁₄H₇F₂NO₃ (MH⁺)276.0467, found 276.0466.

6-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole (17). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 17 as a white solid (7.4 mg, 12%). Data for 17: ¹H NMR (600 MHz,DMSO-d₆) δ 13.20 (br. s, 1H, CO₂H), 8.48 (ABX, 1H, J=7.9 Hz, Ar), 8.41(s, 1H, Ar), 8.28 (ABM, 1H, J=1.5 Hz, Ar), 8.03 (ABX, 1H, J=7.9 Hz, Ar),8.02 (ABM, 1H, J=1.5, 8.2 Hz, Ar), 7.90 (ABM, 1H, J=8.2 Hz, Ar), 7.86(ABX, 1H, J=7.9 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.0, 164.6,151.4, 146.2, 132.8, 132.2, 131.4 (q, J=32 Hz), 130.2, 129.8, 128.4,127.8, 125.2, 125.0 (q, J=272 Hz), 121.2, 113.6; HRMS (MALDI-FTMS)calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0530. HRMS (MALDI-FTMS)calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0531.

6-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole (18). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 18 as a white solid (6.6 mg, 11%). Data for 18: ¹H NMR (600 MHz,DMSO-d₆) δ 13.22 (br. s, 1H, CO₂H), 8.30 (ABX, 1H, J=0.6, 1.5 Hz, Ar),8.20 (d, 1H, J=7.3 Hz, Ar), 8.06 (ABX, 1H, J=1.5, 8.2 Hz, Ar), 8.04 (d,1H, J=7.9 Hz, Ar), 7.98 (ABX, 1H, J=0.6, 8.2 Hz, Ar), 7.94 (T, 1H, J=7.3Hz, Ar), 7.90 (t, 1H, J=7.9 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 168.0,164.0, 151.6, 145.9, 133.8, 130.0, 129.0 (q, J=32 Hz), 128.6 (q, J=6),127.7, 126.0, 124.7 (q, J=273 Hz), 121.6, 113.6; HRMS (MALDI-FTMS)calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0530.

6-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole (19). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 19 as a white solid (6.0 mg, 10%). Data for 19: ¹H NMR (600 MHz,DMSO-d₆) δ 13.20 (br. s, 1H, CO₂H), 8.17 (ABX, 1H, J=0.6, 1.5 Hz, Ar),8.00 (AB, 1H, J=2.0 Hz, Ar), 7.96 (ABX, 1H, J=1.5,8.5 Hz, Ar), 7.83 (AB,1H, J=2.0 Hz, Ar), 7.82 (ABX, 1H, J=0.6, 8.5 Hz, Ar); ¹³C NMR (150 MHz,DMSO-d₆) δ 166.6, 161.9, 150.0, 144.6, 135.1, 131.6, 129.0, 128.7,126.4, 125.8, 119.9, 112.1; HRMS (MALDI-FTMS) calcd. for C₁₄H₇Cl₂NO₃(MH⁺) 307.9876, found 307.9879.

6-Carboxy-2-(2,6-dichlorophe2nyl)-benzoxazole (20). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 20 as a white solid (12.7 mg, 21%). Data for 20: ¹H NMR (500 MHz,DMSO-d₆) δ 13.27 (br. s, 1H, CO₂H), 8.38 (ABX, 1H, J=0.5, 1.5 Hz, Ar),8.09 (ABX, 1H, J=1.5, 8.3 Hz, Ar), 8.02 (ABX, 1H, J=8.3, 0.5 Hz, Ar),7.78–7.71 (m, 3H, Ar); ^(—)C NMR (125 MHz, DMSO-d₆) δ 166.6, 159.8,150.0, 143.8, 134.8, 134.2, 129.1, 128.8, 126.4, 126.3, 120.4, 112.6;HRMS (MALDI-FTMS) calcd. for C₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9877.

6-Carboxy-2-phenyl-benzoxazole (21). Prepared from4-amino-3-hydroxybenzoic acid according to the general procedure, toafford 21 as a white solid (7.0 mg, 15%). Data for 21: ¹H NMR (600 MHz,DMSO-d₆) δ 13.16 (br. s, 1H, CO₂H), 8.27 (d, 1H, J=0.9 Hz, Ar),8.25–8.22 (m, 2H, Ar), 8.01 (dd, 1H, J=1.5, 8.5 Hz, Ar), 7.89 (d, 1H,J=8.5 Hz, Ar), 7.69–7.62 (m, 3H, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ166.8, 164.7, 150.0, 145.2, 132.6, 129.4, 128.0, 127.6, 126.3, 126.0,119.6, 112.0; HRMS (MALDI-FTMS) calcd. for C₁₄H₉NO₃ (MH⁺) 240.0655,found 240.0655.

7-Carboxy-2-(3,5-difluorophenyl)-benzoxazole (22). Prepared from3-aminosalicylic acid according to the general procedure, to afford 22as a white solid (8.8 mg, 16%). Data for 22: ¹H NMR (600 MHz, DMSO-d₆) δ13.55 (br. S, CO₂H), 8.10 (AMX, 1H, J=1.2, 7.9 Hz, Ar), 7.97 (AMX, 1H,J=1.2, 7.9 Hz, Ar), 7.80–7.79 (m, 2H, Ar), 7.63 (tt, 1H, J=2.4, 9.2 Hz,Ar), 7.55 (AMX, 1H, J=7.9 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5,162.8 (d, J=248 Hz), 162.6 (d, J=248 Hz), 160.9, 149.2, 142.6, 129.2,128.0, 125.2, 124.9, 116.1, 110.6 (d, J=28 Hz), 107.7 (q, J=25 Hz); HRMS(MALDI-FTMS) calcd. for C₁₄H₇F₂NO₃ (MH⁺) 276.0467, found 276.0469.

7-Carboxy-2-(2,6-difluorophenyl)-benzoxazole (23). Prepared from3-aminosalicylic acid according to the general procedure, to afford 23as a white solid (7.3 mg, 13%). Data for 23: ¹H NMR (600 MHz, DMSO-d₆) δ13.48 (br. s, 1H, CO₂H), 8.16 (ABX, 1H, J=1.2, 8.2 Hz, Ar), 8.00 (ABX,1H, J=1.2, 7.6 Hz, Ar), 7.78 (m, 1H, Ar), 7.57 (ABX, 1H, J=7.6, 8.2 Hz,Ar), 7.40 (t, 2H, J=8.5 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5,160.4 (d, J=256 Hz), 160.3 (d, J=257 Hz), 154.9, 148.9, 142.1, 134.8 (t,J=10 Hz), 128.0, 125.1, 125.0, 116.0, 113.0 (d, J=22 Hz), 112.9 (q, J=21Hz), 105.1 (t, J=17 Hz); HRMS (MALDI-FTMS) calcd. for C₁₄H₇F₂NO₃ (MH⁺)276.0467, found 276.0467.

7-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole (24). Prepared from3-aminosalicylic acid according to the general procedure, to afford 24as a white solid (7.9 mg, 13%). Data for 24: ¹H NMR (600 MHz, DMSO-d₆) δ13.51 (br. s, CO₂H), 8.48 (ABX, 1H, J=8.2 Hz, Ar), 8.40 (s, 1H, Ar),8.10 (AMX, 1H, J=1.2, 7.9 Hz, Ar), 8.05 (ABX, 1H, J=7.9 Hz, Ar), 7.96(AMX, 1H, J=1.2, 7.6 Hz, Ar), 7.94 (ABX, 1H, J=7.9 Hz, Ar), 7.54 (AMX,1H, J=7.9, 7.6 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.6, 161.7,149.3, 142.7, 131.3, 131.0, 130.0 (q, J=32 Hz), 128.6 (d, J=3 Hz),127.7, 127.2, 125.0, 124.8, 123.7 (q, J=272 Hz), 123.5, 116.0; HRMS(MALDI-FTMS) calcd. for C₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0532.

7-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole (25). Prepared from3-aminosalicylic acid according to the general procedure, to afford 25as a white solid (13.8 mg, 22%). Data for 25: ¹H NMR (600 MHz, DMSO-d₆)δ 13.46 (br. s, 1H, CO₂H), 8.18 (d, 1H, J=7.6 Hz, Ar), 8.14 (AMX, 1H,J=1.2, 7.9 Hz, Ar), 8.03 (d, 1H, J=7.9 Hz, Ar), 7.98 (AMX, 1H, J=1.2,7.6 Hz, Ar), 7.94 (t, 1H, J=7.3 Hz, Ar), 7.89 (t, 1H, J=7.6 Hz, Ar),7.56 (AMX, 1H, J=7.9 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.6, 161.1,149.3, 142.4, 133.0, 132.4, 132.2, 127.8, 127.6, 127.2 (q, J=6 Hz),125.0, 124.9, 123.4 (q, J=273 Hz), 116.2; HRMS (MALDI-FTMS) calcd. forC₁₅H₈F₃NO₃ (MH⁺) 308.0529, found 308.0534.

7-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole (26). Prepared from3-aminosalicylic acid according to the general procedure, to afford 26as a white solid (7.0 mg, 11%). Data for 26: ¹H NMR (600 MHz, DMSO-d₆) δ14.00–12.80 (br. s, CO₂H), 8.10–8.08 (m, 3H, Ar), 7.98–7.96 (m, 2H, Ar),7.55 (t, 1H, J=7.8 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.5, 160.5,149.2, 142.6, 135.2, 131.5, 129.4, 128.0, 125.6, 125.2, 124.8, 116.2;HRMS (MALDI-FTMS) calcd. for C₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9874.

7-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole (27). Prepared from3-aminosalicylic acid according to the general procedure, to afford 27as a white solid (10.3 mg, 17%). Data for 27: ¹H NMR (600 MHz, DMSO-d₆)δ 13.90–13.10 (br. s, CO₂H), 8.16 (AMX, 1H, J=7.9 Hz, Ar), 8.02 (AMX,1H, J=7.9 Hz, Ar), 7.78–7.72 (m, 3H, Ar), 7.60 (AMX, H, J=7.9 Hz, Ar);¹³C NMR (150 MHz, DMSO-d₆) δ 164.4, 158.3, 149.1, 141.7, 134.9, 134.2,128.8, 128.2, 126.5, 125.3, 125.2, 116.2; HRMS (MALDI-FTMS) calcd. forC₁₄H₇Cl₂NO₃ (MH⁺) 307.9876, found 307.9875.

7-Carboxy-2-phenyl-benzoxazole (28). Prepared from 3-aminosalicylic acidaccording to the general procedure, to afford 28 as a white solid (13.1mg, 27%). Data for 28: ¹H NMR (600 MHz, DMSO-d₆) δ 13.48 (br. s, 1H,CO₂H), 8.20–8.19 (m, 2H, Ar), 8.05 (AMX, 1H, J=1.2, 7.9 Hz, Ar), 7.92(AMX, 1H, J=1.2, 7.6 Hz, Ar), 7.66–7.62 (m, 3H, Ar), 7.50 (AMX, 1H,J=7.9 Hz, Ar); ¹³C NMR (150 MHz, DMSO-d₆) δ 164.8, 163.1, 149.2, 142.9,132.2, 129.4, 127.4, 127.2, 126.0, 124.7, 124.4, 115.8; HRMS(MALDI-FTMS) calcd. for C₁₄H₉NO₃ (MH⁺) 240.0655, found 240.0656.

OTHER EMBODIMENTS

It is to be understood that, while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications of the inventionare within the scope of the claims set forth below.

1. A method of treating a transthyretin amyloid disease, comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of a compound of formula

or a pharmaceutically acceptable salt thereof, wherein Ar is phenyl,3,5-difluorophenyl, 2,6-difluorophenyl, 3,5-dichlorophenyl,2,6-dichlorophenyl, 2-(trifluoromethyl)phenyl, or3-(trifluoromethyl)phenyl.
 2. The method of claim 1, wherein thecompound is: 4-Carboxy-2-(3,5-difluorophenyl)-benzoxazole;4-Carboxy-2-(2,6-difluorophenyl)-benzoxazole;4-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole;4-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole;4-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole;4-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole;4-Carboxy-2-phenyl-benzoxazole;5-Carboxy-2-(3,5-difluorophenyl)-benzoxazole;5-Carboxy-2-(2,6-difluorophenyl)-benzoxazole;5-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole;5-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole;5-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole;5-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole;5-Carboxy-2-phenyl-benzoxazole;6-Carboxy-2-(3,5-difluorophenyl)-benzoxazole;6-Carboxy-2-(2,6-difluorophenyl)-benzoxazole;6-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole;6-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole;6-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole;6-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole;6-Carboxy-2-phenyl-benzoxazole;7-Carboxy-2-(3,5-difluorophenyl)-benzoxazole;7-Carboxy-2-(2,6-difluorophenyl)-benzoxazole;7-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole;7-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole;7-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole;7-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole; or7-Carboxy-2-phenyl-benzoxazole.
 3. The method of claim 1, wherein thecompound is 6-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole.
 4. The methodof claim 1, wherein the transthyretin amyloid disease is familialamyloid polyneuropathy.
 5. The method of claim 2, wherein thetransthyretin amyloid disease is familial amyloid polyneuropathy.
 6. Themethod of claim 3, wherein the transthyretin amyloid disease is familialamyloid polyneuropathy.
 7. The method of claim 1, wherein thetransthyretin amyloid disease is familial amyloid cardiomyopathy.
 8. Themethod of claim 2, wherein the transthyretin amyloid disease is familialamyloid cardiomyopathy.
 9. The method of claim 3, wherein thetransthyretin amyloid disease is familial amyloid cardiomyopathy. 10.The method of claim 1, wherein the transthyretin amyloid disease issenile systemic amyloidosis.
 11. The method of claim 2, wherein thetransthyretin amyloid disease is senile systemic amyloidosis.
 12. Themethod of claim 3, wherein the transthyretin amyloid disease is senilesystemic amyloidosis.
 13. The method of claim 1, wherein thetransthyretin amyloid disease is cardiac amyloidosis following livertransplantation.
 14. The method of claim 2, wherein the transthyretinamyloid disease is cardiac amyloidosis following liver transplantation.15. The method of claim 3, wherein the transthyretin amyloid disease iscardiac amyloidosis following liver transplantation.
 16. The method ofclaim 1, wherein the compound is5-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole.
 17. The method of claim16, wherein the transthyretin amyloid disease is familial amyloidpolyneuropathy.
 18. The method of claim 16, wherein the transthyretinamyloid disease is familial amyloid cardiomyopathy.
 19. The method ofclaim 16, wherein the transthyretin amyloid disease is senile systemicamyloidosis.
 20. The method of claim 16, wherein the transthyretinamyloid disease is cardiac amyloidosis following liver transplantation.21. The method of claim 1, wherein the compound is4-Carboxy-2-(3,5-difluorophenyl)-benzoxazole.
 22. The method of claim 1,wherein the compound is 4-Carboxy-2-(2,6-difluorophenyl)-benzoxazole.23. The method of claim 1, wherein the compound is4-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole.
 24. The method ofclaim 1, wherein the compound is4-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole.
 25. The method ofclaim 1, wherein the compound is4-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole.
 26. The method of claim 1,wherein the compound is 4-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole.27. The method of claim 1, wherein the compound is4-Carboxy-2-phenyl-benzoxazole.
 28. The method of claim 1, wherein thecompound is 5-Carboxy-2-(3,5-difluorophenyl)-benzoxazole.
 29. The methodof claim 1, wherein the compound is5-Carboxy-2-(2,6-difluorophenyl)-benzoxazole.
 30. The method of claim 1,wherein the compound is5-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole.
 31. The method ofclaim 1, wherein the compound is5-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole.
 32. The method ofclaim 1, wherein the compound is5-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole.
 33. The method of claim 1,wherein the compound is 5-Carboxy-2-phenyl-benzoxazole.
 34. The methodof claim 1, wherein the compound is6-Carboxy-2-(3,5-difluorophenyl)-benzoxazole.
 35. The method of claim 1,wherein the compound is 6-Carboxy-2-(2,6-difluorophenyl)-benzoxazole.36. The method of claim 1, wherein the compound is6-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole.
 37. The method ofclaim 1, wherein the compound is6-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole.
 38. The method ofclaim 1, wherein the compound is6-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole.
 39. The method of claim 1,wherein the compound is 6-Carboxy-2-phenyl-benzoxazole.
 40. The methodof claim 1, wherein the compound is7-Carboxy-2-(3,5-difluorophenyl)-benzoxazole.
 41. The method of claim 1,wherein the compound is 7-Carboxy-2-(2,6-difluorophenyl)-benzoxazole.42. The method of claim 1, wherein the compound is7-Carboxy-2-[(3-trifluoromethyl)phenyl]-benzoxazole.
 43. The method ofclaim 1, wherein the compound is7-Carboxy-2-[(2-trifluoromethyl)phenyl]-benzoxazole.
 44. The method ofclaim 1, wherein the compound is7-Carboxy-2-(3,5-dichlorophenyl)-benzoxazole.
 45. The method of claim 1,wherein the compound is 7-Carboxy-2-(2,6-dichlorophenyl)-benzoxazole.46. The method of claim 1, wherein the compound is7-Carboxy-2-phenyl-benzoxazole.